Method for measuring substance and testing piece

ABSTRACT

A method of measuring an analyte, comprising a step of measuring a detectable substance by using a reaction system including a formation reaction of the detectable substance based on a chemical reaction of the analyte contained in a sample, wherein a layered inorganic compound is caused to exist in the reaction system including the formation reaction of the detectable substance, whereby high-sensitivity measurement is made possible, the detectable substance can be stabilized to improve accuracy of the measurement, a rate of a chemical reaction is increased to enable quick measurement, and high-sensitivity measurement is made possible even in a reaction system which forms an insoluble substance. Also, it can be provided an analytical testing piece for measuring an analyte, by measuring a detectable substance by using a reaction system including a formation reaction of the detectable substance based on a chemical reaction of the analyte contained in a sample wherein the testing piece comprises at least one test portion having a detection portion for detecting the detectable substance and contains a layered inorganic compound at least in the test portion, whereby diffusion and elution of a dyestuff or the like is prevented, more sensitive and accurate simple analysis is made possible, and easy handling is possible.

FIELD OF THE INVENTION

[0001] The present invention relates to a method for measuring ananalyte such as a biological component or environmental substance byusing a reaction system which forms a detectable substance such as adyestuff based on the chemical reaction of the analyte contained in asample and measuring the detectable substance, and to a testing piecefor use in the method.

BACKGROUND OF THE INVENTION

[0002] Methods of detecting and determining an analyte which iscontained in a sample, for example, a bio-component in the body fluidsuch as urine and blood, a trace amount of a substance existent in food,medicine, or natural environment, an industrial chemical substance, atrace amount of a substance contained in waste, or the like include onesfor measuring a detectable substance such as a dyestuff formed by areaction system in which the analyte is involved.

[0003] One of the methods is, for example, a method comprisingsubjecting hydrogen peroxide formed by the chemical reaction of theanalyte and a reactive color coupler (dyestuff precursor) to anoxidation-reduction reaction in the presence of peroxidase (POD) anddetermining the formed dyestuff compound by colorimetry. This method isfrequently used in clinical diagnosis and the like because of itssimplicity. Another one of the methods is a method of measuring ananalyte based on an electrochemical reaction for reducing/oxidizing withan electrode the oxidized/reduced form of an electron carrier (mediator)formed by an oxidation-reduction reaction between the electron carrierand the analyte caused by an enzyme or the like.

[0004] However, in the above conventional methods, as measurementsensitivity is not sufficiently high when the amount of the analyte isvery small, a highly accurate measurement result cannot be obtained.Therefore, the development of a highly accurate measuring method havingimproved measurement sensitivity has been desired.

[0005] Further, since measurement takes long as a reaction takes time,or it takes time for a detection reaction to reach a termination, arating method for carrying out quantitative determination from areaction rate has such a problem that the accuracy of quantitativedetermination is low. To cope with this, to increase the reaction rate,the reaction system is heated, or the concentration of a reagent for thereaction is increased. However, in the method for heating the reactionsystem, a heat source is required for heating and analysis is therebycomplicated. When the formed substance is thermally instable, detectionis difficult and this means cannot be employed. The method forincreasing the concentration of the reagent is not practical because itleads to a rise in the background of detection and an increase in thecost of analysis. There is also a method for adding a catalyst toincrease the reaction rate. However, since there are many detectionreactions for which preferred catalysts are unknown yet, this method isnot practical as well. As described above, most of the conventionalmethods are still unsatisfactory and a novel method which enables quickmeasurement by increasing the reaction rate more simply has been eagerlydesired.

[0006] When a reaction which forms a substance insoluble in a reactionsolvent is included in the reaction system which forms a detectablesubstance, there is such inconvenience as enumerated below and called inquestion.

[0007] (1) In measurement in which optical detection is carried outusing a liquid reagent, for example, in a batch type automaticbiochemical test apparatus, when a dyestuff formed by a reaction isinsoluble in a solvent, it separates out and adheres to the wall of ameasurement cell to shield incident light or transmitted light or causethe pollution of a dispensing nozzle, and abnormality in absorptioncoefficient, diffusion or light-shielding by agglomeration, therebymaking measurement difficult.

[0008] (2) Similarly, in measurement in which optical detection iscarried out using a liquid reagent, when an insoluble by-product isformed, it adheres to the wall of a measurement cell to shield incidentlight or transmitted light or cause the pollution of a dispensingnozzle, and diffusion or light shielding by agglomeration, therebymaking measurement difficult.

[0009] (3) In measurement in which a dyestuff formed by a reactioncaused by dropping onto or infiltrating a sample to be measured into atesting piece is optically detected, when the formed dyestuff isinsoluble in a sample solvent, the dyestuff deposits on the substrate ofthe testing piece nonuniformly, or the agglomeration of the dyestuffoccurs, thereby deteriorating measurement accuracy.

[0010] (4) In electrode measurement using a liquid reagent, for example,in a batch type automatic biochemical test apparatus, when an insolubleby-product is formed, the pollution of an electrode is caused bycovering the surface of the electrode with the insoluble deposit,thereby reducing biochemical response and deteriorating measurementaccuracy.

[0011] The difference between the words “insoluble” and “hardly soluble”indicates a difference in the degree of insolubility in a solvent. Inthe present invention, the word “insoluble” may be interchanged by“hardly soluble” in the following description.

[0012] Particularly, when the formed detectable substance is insolublein a reaction solvent, reaction rate may be reduced, or measurementsensitivity may be lowered because the reaction system which forms thedetectable substance is not uniform and the reaction does not proceedquickly in the prior art method. For example, in the reaction systemusing an enzyme, the reaction product may deposit near the enzyme orimpede the reaction.

[0013] Therefore, a measuring method using a reaction which forms aby-product insoluble in a reaction solvent in which the reaction iscarried out has rarely been employed. Accordingly, it has been necessaryto select a reaction which does not form an insoluble product as adetection system or to develop a new detection reaction system bysynthetic chemical means so that the product becomes soluble in areaction solvent. However, these circumstances have limited a reactionsystem used. Meanwhile, much time and labor have been required for theresearch and development of a reaction system which forms only a solublesubstance. Further, it has been necessary to add a surfactant forsolubilizing, emulsifying or dispersing the product. However, theaddition of a surfactant is disadvantageous from the view point ofmeasurement cost and may produce an adverse effect such as interruptionof a reaction. Therefore, it cannot be said that it is a perfectsolution. Then, a novel method which solves this problem easily andenables measurement in the presence of an insoluble product has beenardently desired.

[0014] The method of measuring an analyte using a reaction system whichforms hydrogen peroxide as described above is an important measuringmethod as there are many reactions which form hydrogen peroxide as asubstance produced by oxidation. However, accurate measurement has notalways been easy in the prior art methods for the following reasons.That is, in these measuring methods, the amount or concentration of adetectable substance such as a dyestuff compound must have aquantitative correlation with a specific substance such as hydrogenperoxide in some cases. However, an oxidation-reduction system incolorimetry is affected by the strong oxidizing activity of excessivehydrogen peroxide or the strong reducing activity of ascorbic acid orthe like contained in a biological sample, and the above detectablesubstance such as a dyestuff compound decomposes, whereby a measurementerror may be produced.

[0015] For example, in these measuring methods, when an excessive amountof hydrogen peroxide is temporarily formed from an analyte such asglucose by an oxidase such as glucose oxidase, a reaction between theformed dyestuff and hydrogen peroxide occurs in addition to a reactionbetween a dyestuff precursor and hydrogen peroxide. As a result, theformed dyestuff is decomposed by hydrogen peroxide as soon as it isformed and discolored.

[0016] When an enzyme such as peroxidase for producing active oxygenspecies such as a superoxide having high reactivity from hydrogenperoxide, or transition metal ions and a complex thereof exerting asimilar function are existent in a sample, the active oxygen speciesreact with the formed dyestuff, decomposes and discolors it. Thisinterference has affected measurement adversely. When a reaction whichforms a detectable substance such as a dyestuff is carried out while itis exposed to the air, the formed dyestuff may be oxidized by oxygencontained in the air or oxygen dissolved in a reaction solution,decomposed and discolored.

[0017] Therefore, various attempts have been made such as the researchof a dyestuff precursor which provides a stable substance which ishardly decomposed and the addition of various stabilizers but these arestill unsatisfactory.

[0018] Reducing substances such as ascorbic acid, uric acid andbilirubin contained in a biological sample have a great influence on anoxidation-reduction reaction. Particularly, how to measure an analyteaccurately in the presence of ascorbic acid has been a significant themein the field of clinical analysis for long time. Various interferencesuppression means such as selective decomposition with an enzyme,decomposition by the addition of periodic acid, oxidation decompositionwith iron-ethylene diamine tetraacetate chelate, and selectiveseparation with a semipermeable membrane have been tried in addition tothe research of the above-described dyestuff precursor and the like (seeYoshihide Ohta, Yutaka Ogawa, Rinsho Kensa, 34 (4), 502-504 (1990);Japanese Patent Publication No. 1-41223(1989); Japanese PatentPublication No. 2-4861(1990); Japanese Patent Publication No.4-18630(1992); Japanese Patent Application Laid-open No. 5-95797(1993);and Japanese Patent Application Laid-open No. 7-155196(1995)).

[0019] There are further methods of measuring a specific analyte byforming a dyestuff (for example, an azo dyestuff) having quantitativerelationship with the specific analyte by various known reactions otherthan the oxidation-reduction reaction (for example, condensationreactions such as an acid-base reaction and the coupling reaction of adiazonium salt, a complex forming reaction and the like) and opticallydetermining the formed dyestuff. These methods are important measuringmethods detailed in Bunseki Kagaku Binran (ed. by the Japan Society forAnalytical Chemistry), for example. However, some of the thus formeddyestuffs may be an instable compound which is decomposed by oxygen inthe atmosphere, an oxidizing or reducing substance contained in asample, hydrogen ions or bases contained in the sample, light or thelike. To measure this substance, for example, quick operation isrequired, or operation must be carried out in an atmosphere substitutedby nitrogen or light shaded environment. Otherwise, an error may begiven to measurement.

[0020] Methods using an electron carrier (mediator) include one in whichan analyte is measured with high sensitivity by carrying out an enzymereaction for a predetermined time to oxidize/reduce the electron carrierduring that time, thereby accumulating the oxidized/reduced form of theelectron carrier, and reducing/oxidizing the accumulatedoxidized/reduced form of the electron carrier with an electrode afterthe predetermined time to produce great electrochemical response.Conventionally, the accumulated oxidized/reduced form of the electroncarrier has been subjected to a decomposition reaction such asreduction/oxidation by a reducing substance or oxidizing substance whichis coexistent with the accumulated oxidized/reduced form of the electroncarrier, whereby an error may be given to measurement.

[0021] When the detectable substance is stable without being decomposed,the quantitative relationship is ensured at the time of measurement anda more excellent S/B ratio (signal-to-background ratio) can be obtainedby carrying out time integration, whereby the accuracy of analysis canbe improved and sensitivity can be increased. Therefore, to develop areaction system which forms a detectable substance which is stable andcan be-measured easily, many efforts have been made so far. Variousreagents which have been developed so far as reaction substances whichform such a stable detectable substance are listed in many handbooks,Bunseki Kagaku Binran, for example.

[0022] However, the research of a reaction system which forms such astable substance takes much time and labor, and efforts are still beingmade to search for a reaction system which forms a detectable substancewhich is always stable and can be measured easily. Therefore, even incurrently used measurement methods, there are many cases where aninstable substance which is decomposed by pH, moisture content,coexistent substance such as an oxidizing/reducing substance, light orthe like must be measured as the detectable substance.

[0023] An analytical testing piece, used to examine and analyze acomponent contained in a liquid sample such as urine, for measuring ananalyte by measuring a detectable substance such as a formed dyestuffbased on the chemical reaction of the analyte contained in a sample,generally comprises a test portion which is a functional portion forcarrying out a series of analytical processes such as the absorption,diffusion, reaction, detection and the like of the liquid sample and asupport portion for supporting the test portion, and further has asensor, sample solution suction apparatus and the like as required. Theabove test portion comprises layers or areas for carrying out variousfunctions. Generally speaking, the test portion comprises a samplesuction portion for sucking the sample and introducing it thereinto; adiffusion and infiltration portion for diffusing and infiltrating thesample uniformly in the test portion; a reagent portion containing areagent which reacts with the analyte contained in the sample; areaction portion where a reaction such as a detection reaction occurs; adeveloping portion for separating a component contained in the sample, adyestuff formed by the detection reaction or the like by achromatography-like function such as adsorption or distribution; a timecontrol portion for adjusting the proceeding of a reaction making use ofa time during which the sample moves; a holding portion for trapping orremoving a component contained in the sample, formed dyestuff or thelike by an adsorption function; a detection portion for detecting adyestuff or the like by reflectance, transmission/absorption orfluorescence; an absorbing portion for absorbing excess of a samplesolution, added washing solution and developing solution to prevent aback flow, and the like.

[0024] In an actual testing piece, these portions having the abovefunctions are not always existent independently. For example, likelitmus paper in which the detection portion is the same as the samplesuction portion, the reagent portion and the reaction portion, there isa case where one portion has multiple functions.

[0025] For example, there are single-layered and multi-layered testingpieces which comprise a diffusion layer which also serves as a samplesuction layer, a detection layer which also serves as a reagent layerand a reaction layer, or comprise a detection layer independent from areaction layer which also serves as a reagent layer. Most of them arebonded to a base by an adhesive layer. There is a testing piece whichhas a developing layer or a holding layer having a function to remove aninterfering component between a reaction layer and a detection layer.There is also a testing piece in which a diffusion layer also serves asa developing layer and is in contact with a reagent layer by an adhesivelayer. When detection is carried out by measuring reflectance, areflection layer may be formed before or after a detection layer. Thesample is dropped onto the diffusion layer which also serves as thesample suction layer and diffused uniformly to dissolve a reagentcontained in the reagent layer, whereby a reaction proceeds. Thus, forexample, a dyestuff is produced from a dyestuff precursor. When thereagent layer and the reaction layer also serve as the detection layer,the dyestuff is directly measured. However, when an independentdetection layer is provided, the produced dyestuff or the like furtherinfiltrates and moves into the detection layer and is measured at thatpoint (see H. G. Curme, et al., Clinical Chemistry, 24 (8), 1335-1342(1978); B. Walter, Analytical Chemistry, 55 (4), 498A (1983); AsajiKondo, Bunseki, 1984 (7), 534; Asaji Kondo, Bunseki, 1986 (6), 387;Bunseki Kagaku Binran, p. 8 (edited by the Japan Society for AnalyticalChemistry: fourth revised edition, Maruzen (1991); and Japanese PatentApplication Laid-open No. 6-213886(1994) (Masao Kitajima et al.)).

[0026] There is also a testing piece which comprises an infiltrationportion of a developing solution at an end of the testing piece on asmall piece of filter paper; a sample suction portion adjacent to theinfiltration portion; a reaction portion which also serves as a reagentportion (having an enzyme immobilized thereto) near the center of thetesting piece; and a detection portion which also serves as a reagentportion (having a dyestuff precursor or the like immobilized thereto), areaction portion and a holding portion after the reaction portion andmakes use of the plane movement of the sample or the like. In this case,after the sample is dropped onto the sample suction portion, thedeveloping solution is infiltrated from the end of the testing piece tomove the sample by a capillary action, the sample reacts with the enzymein the reaction portion which also serves as the first reagent portion(having the enzyme immobilized thereto) to produce hydrogen peroxidewhich is then moved by the developing solution to color the dyestuffprecursor or the like in the detection portion which also serves as thesecond reagent portion (having the dyestuff precursor or the likeimmobilized thereto), the reaction portion and the holding portion, andadsorb and hold the produced dyestuff or the like (detectablesubstance). Since the hydrogen peroxide moves along with the movement ofthe developing solution and a coloration reaction occurs along with themovement, when the amount of the analyte increases, the length ofcoloration expands, whereby the substance can be measured. (see M. P.Allen, et al., Clinical Chemistry, 36 (9), 1591-1597 (1990); D. Noble,Analytical Chemistry, 65 (23), 1037A (1993).)

[0027] This testing piece is used in a urine test, a biochemical test,an immunochromatography test and the like. In an example of a testingpiece for immunochromatography, when one end of filter paper having anantibody immobilized thereto (it can be said that the entire surfacethereof serves as a reagent portion, a reaction portion, a developingportion, a holding portion and a detection portion) is immersed in adeveloping solution prepared by mixing a sample containing an antigen(analyte) and an enzyme-linked antigen as a reagent to develop with acolor developing solution which is a second reagent (containing adyestuff precursor), a portion containing the enzyme-linked antigenwhich has been developed and captured is colored like a belt. The lengthof the colored belt is proportional to the amount of the antigencontained in the sample. (see R. F. Zuk, et al., Clinical Chemistry, 31(7), 1144-1150 (1985).)

[0028] As another example of a testing piece for immunochromatography,there is a testing piece which comprises a reagent portion (firstantibody immobilized colored latex) which also serves as a samplesuction portion at one end on a small piece of a membrane filter, areagent portion (second antibody which recognizes the same antigen asthat of the first antibody but is different in epitope) which alsoserves as a developing portion near the center, a developing portion andfurther a detection portion which also serves as a reagent portion(anti-first antibody antibody) and a holding portion. When a sample isdropped onto the sample suction portion, an antigen-antibody reactionbetween an antigen (analyte) and the first antibody occurs, animmuno-complex directly moves along with the movement of the sample, anda sandwich reaction between the immuno-complex and the second antibodyoccurs in the reagent portion which also serves as a developing portion.However, excess of the first antibody which does not form animmuno-complex passes through the developing portion along with themovement of the sample and is captured in the detection portion whichalso serves as the reagent portion (anti-first antibody antibody) andthe holding portion. The analyte can be measured by measuring thecoloration of the colored latex (containing a dyestuff as a detectablesubstance) to which the first antibody is immobilized. (see I. W.Davidson, Analytical Proceedings, 29, 459 (1992).)

[0029] However, in the above testing pieces, a dyestuff or the likeproduced by a reaction with a component to be analyzed has solubility ina sample solution, reaction solution or the like in many cases with theresult of such inconvenience as the elution of the dyestuff or the likeinto a bulk solution, a back flush to the diffusion layer, and theadhesion of the dyestuff or the like to the adjacent test portion inmulti-item test paper having a plurality of test portions. Due to themovement of the dyestuff or the like toward the edge of the test portionby drying, there occurs such a phenomenon that the concentration of acenter portion becomes low and that of a peripheral portion becomeshigh.

[0030] Such an inconvenient phenomenon that deteriorates measurementsensitivity, precision and accuracy is particularly marked in urine testpaper or the like which is immersed in a sample solution for measurementbut is very common irrespective of the type of sample.

[0031] Meanwhile, there have been proposed a method for preventing theelution of a reagent by covering a test portion (Japanese PatentApplication Laid-open No. 2-38861(1990)), a method for preventing liquidjunction between adjacent test portions by causing the test portionscomposed of a porous structure (such as a porous layer or a porous film)having high absorptivity to uniformly absorb a sample (Japanese PatentApplication Laid-open No. 2-6541(1990)), a method for selecting areaction for forming an insoluble dyestuff, a method for capturing aformed dyestuff using an insoluble and hydrophobic binder (fixing agent)(Japanese Patent Application Laid-open No. 7-181174(1995)), a method forincreasing the distance between adjacent test portions in the multi-itemtest paper, a method for controlling and adjusting immersion time, amethod for controlling time so that measurement is carried out beforediffusion, and the like. However, covering a test portion or preparing aporous structure by a precipitation-solidification method makes a testpaper production process complicated. When a reaction for forming aninsoluble dyestuff is selected, a product inhibition of enzyme activityoccurs. A testing piece prepared by using a hydrophobic polymer as abinder has such a defect that the absorptivity of an aqueous samplesolution deteriorates. A multi-item testing piece has such a defect thatwhen the distance between adjacent test portions is increased, a largerarea is required or it is disadvantageous for the movement of the sensoras a single sensor moves through a plurality of test portions to measurereflected light. The other methods have respective problems to besolved. For example, the method for controlling immersion time istroublesome in an urine test, the method for controlling time i's noteasy because of the relationship between control time and reaction time.Satisfactory solutions to these problems are yet to be found.

[0032] A method for measuring an analyte from electrochemical responseat the time of oxidation-reduction using the above electron carrier(mediator), a method for measuring ions as an analyte by measuring thepotential of a membrane upon the movement of a complex compound formedby using a ligand (ionophore) which is coordinately bonded or ion bondedto a specific ion in a liquid film electrode, and the like are known asimportant measuring methods. Generally speaking, in an electrodecomposed of an oxidized/reduced form of an electron carrier or a complexcompound, the elution or diffusion of the electron carrier or ligand isprevented by adding the electron carrier or ligand to an insolublepolymer, and the electron carrier or ligand is held near the surface ofthe electrode so that electrons can move quickly at the same time. Sincethe movement of a substance in a polymer is limited, a reaction betweenan analyte contained in the sample or an intermediate substance producedfrom the analyte and the electron carrier or ligand contained in theinsoluble polymer is interrupted. A satisfactory solution to thisfundamental problem is yet to be found as well.

DISCLOSURE OF THE INVENTION

[0033] It is an object of the present invention to provide ahigh-sensitivity measuring method for measuring an analyte by measuringa detectable substance such as a dyestuff or the like formed based onthe chemical reaction of the analyte. The term “measurement” comprehendsboth quantitative and qualitative measurements.

[0034] It is another object of the present invention to provide a methodwhich can improve measurement accuracy and increase measurementsensitivity by stabilizing the detectable substance in the above methodfor measuring the analyte.

[0035] It is still another object of the present invention to provide anovel method which enables quick measurement by increasing the reactionrate of a chemical reaction in the above measuring method.

[0036] It is a further object of the present invention to provide ahigh-sensitivity measuring method in the above method using a reactionsystem including the formation reaction of an insoluble substance.

[0037] It is a still further object of the present invention to providean analytical testing piece which can suppress the diffusion and elutionof a dyestuff or the like, enables accurate examination and analysis,and is easy to use.

[0038] The inventors of the present invention have found that the aboveproblems can be solved by carrying the formation reaction of adetectable substance in the presence of a layered inorganic compound andby allowing a layered inorganic compound to be contained in a testportion, such as a detection portion for detecting a detectablesubstance, of a testing piece. The present invention has beenaccomplished based on the above finding.

[0039] Thus, the present invention provides a method for measuring ananalyte, comprising a step of measuring a detectable substance by usinga reaction system including a formation reaction of a detectablesubstance based on a chemical reaction of the analyte contained in asample, wherein a layered inorganic compound is caused to exist in thereaction system including the formation reaction of the detectablesubstance. This method will be referred to as “measuring method of thepresent invention” hereinafter.

[0040] The present invention also provides the above-mentioned methodfor measuring the substance comprising a step of adding the layeredinorganic compound to the reaction system to allow the layered inorganiccompound to adsorb the detectable substance. This method will bereferred to as “first method of the present invention” hereinafter.

[0041] In the first method of the present invention, high-sensitivitymeasurement is made possible by allowing the layered inorganic compoundto adsorb the formed detectable substance. That is, for example, thedetectable substance is adsorbed to the layered inorganic compound andsettles, whereby measurement sensitivity in optical or electrochemicaldetection is improved. In this case, the detectable substance may beadsorbed to the layered inorganic compound and settle as a colloidalagglomerate. However, it does not always need to be agglomerated.

[0042] The present invention also provides the method for measuring theanalyte, wherein the layered inorganic compound is caused to exist inthe reaction system to suppress the decomposition of the detectablesubstance. This method will be referred to as “second method of thepresent invention” hereinafter.

[0043] In the second method of the present invention, by causing thelayered inorganic compound to exist in the reaction system which formsthe detectable substance to be measured, a complex between thedetectable substance and the layered inorganic compound is formed almostat the same time when the detectable substance is formed or before it isdecomposed by a coexistent substance with the result that thedecomposition of the detectable substance by the function of thecoexistent substance in the reaction system can be suppressed.

[0044] The present invention further provides the method for measuringthe analyte, wherein the formation reaction of the detectable substanceis carried out in the presence of the layered inorganic compound toincrease a reaction rate of the formation reaction. This method will bereferred to as “third method of the present invention” hereinafter.

[0045] In the third method of the present invention, by carrying out theformation reaction of the detectable substance in the presence of thelayered inorganic compound, the reaction rate of the formation reactionis increased and quick measurement is made possible, thereby greatlyshortening measurement time and also a time required for the detectionreaction to reach a termination with the result that the determinationaccuracy of a rating method for quantity determination from a reactionrate can be improved. The reason for an increase in the rate of theformation reaction of the detectable substance is not always clear butit is considered that the reaction rate is increased by the adsorptionof a reaction starting substance or a reaction intermediate of theformation reaction to the surface of the layered inorganic compound andthe concentration thereof on the surface.

[0046] The present invention further provides the method for measuringthe analyte wherein at least one of reactions constituting the reactionsystem is the formation reaction of a substance insoluble in a reactionsolvent. This method will be referred to as “fourth method of thepresent invention” hereinafter.

[0047] In the fourth method of the present invention, it is possible tomake a reaction proceed quickly like a uniform system by causing alayered inorganic compound to exist in the reaction system including theformation reaction of the detectable substance preferably in a dispersedstate even when the detectable substance or a by-product of the reactionis insoluble in a reaction solvent. It is considered that this isbecause the formed insoluble detectable substance or the insolubleby-product is adsorbed to the layered inorganic compound and uniformlydispersed in the reaction system together with the layered inorganiccompound. In the present invention, the detectable substance or theby-product can be prevented from separating out into the reaction systemand becoming difficult to be handled at the time of detection byallowing the detectable substance or the by-product insoluble in asolvent to be adsorbed by the layered inorganic compound.

[0048] Cases where the detectable substance or the by-product areprevented from becoming difficult to be handled at the time of detectionmay be as follows.

[0049] (1) In measurement in which optical detection is carried outusing a liquid reagent, for example, in a batch type automaticbiochemical test apparatus, when a dyestuff formed by a reaction isinsoluble in a solvent, by allowing the dyestuff to be adsorbed by thelayered inorganic compound, it is possible to prevent the dyestuff fromseparating out and adhering to the wall of a measurement cell to shieldincident light or transmitted light and cause the pollution of adispensing nozzle and abnormality in absorption coefficient, scatteringor light shielding. Thus, it is possible to prevent measurement frombecoming difficult.

[0050] (2) Similarly, in measurement in which optical detection iscarried out using a liquid reagent, when an insoluble by-product isformed, by allowing the by-product to be adsorbed by the layeredinorganic compound, it is possible to prevent the by-product fromadhering to the wall of a measurement cell to shield incident light ortransmitted light and cause the pollution of a dispensing nozzle andscattering or light shielding by agglomeration. Thus, it is possible toprevent measurement from becoming difficult.

[0051] (3) In measurement in which a reaction is carried out by droppingonto or infiltrating a sample to be measured into a testing piece andthe formed dyestuff is optically detected, when the formed dyestuff isinsoluble in a sample solvent, by allowing the dyestuff to be adsorbedby the layered inorganic compound, it is possible to prevent thedyestuff from nonuniformly depositing on the reaction portion or thedetection portion of the testing piece and from being agglomerated,thereby eliminating deterioration in measurement accuracy.

[0052] (4) In electrode measurement using a liquid reagent, for example,in a batch type automatic biochemical test apparatus, when an insolubleby-product is formed, by allowing the by-product to be adsorbed by thelayered inorganic compound, it is possible to prevent the insolubledeposit from covering the surface of the electrode to cause thepollution of the electrode and lower electrochemical response, therebyeliminating deterioration in measurement accuracy.

[0053] A measuring method which the present invention is applied to isnot particularly limited if it is a method for measuring an analyte bymeasuring a detectable substance by using a reaction system includingthe formation reaction of the detectable substance based on the chemicalreaction of the analyte contained in a sample. The detectable substancemay be the analyte as a matter of course. Further, the method may be amethod for measuring an analyte qualitatively by measuring a detectablesubstance or a method for measuring an analyte quantitatively by using areaction system including the formation reaction of a detectablesubstance having a quantitative correlation with the analyte. Moreover,not only a case where a reaction system which forms a detectablesubstance directly by the chemical reaction of an analyte is used butalso a case where the chemical reaction of the analyte and the formationreaction of the detectable substance are indirectly connected to eachother through another chemical reaction are included. Out of thesemethods, the method of the present invention is preferably applied to ameasuring method using a reaction system in which the detectablesubstance is a dyestuff or electron carrier formed by anoxidation-reduction reaction, a measuring method using a reaction systemin which the formed detectable substance is a dyestuff such as an azodyestuff or a complex between an ionophore and an analyte, and the like.

[0054] Particularly, a method of optically measuring a dyestuff formedquantitatively by an oxidation-reduction reaction between hydrogenperoxide formed from a biological component by an oxidizing enzymereaction and a reactive color-producing reagent is used in thequantitative determination of each component contained in the body fluidin clinical examination, environmental analysis and the like. Byapplying the measuring method of the present invention in theseanalytical and detection methods, highly sensitive measurement is madepossible.

[0055] Speaking of the second method of the present invention inparticular, in an oxidation-reduction reaction system, for example, anoxidizing substance, a reducing substance or a peroxidase-like substanceoften exists in the reaction system as a reaction intermediate or animpurity in a sample, and a detectable substance may be decomposed bythe function of these existent substances in the reaction system. Inthis case, the second method of the present invention is useful.

[0056] According to the second method of the present invention, in theabove measuring method using an oxidation-reduction reaction betweenhydrogen peroxide and a reactive color-producing reagent, such a problemthat a measurement error is made by the decomposition and discolorationof a dyestuff or the like caused by the function of an oxidizingsubstance such as excessive hydrogen peroxide or a reducing substancesuch as ascorbic acid, uric acid and bilirubic acid existent in thereaction system can be overcome.

[0057] The third method of the present invention makes it possible toadsorb a reaction starting substance or a reaction intermediate to thesurface off a layered inorganic compound by adding the layered inorganiccompound having cationic exchange ability to a reaction systemparticularly when the starting substance or the intermediate of theformation reaction of a detectable substance is a cationic compound,whereby the formation reaction rate can be improved and quickmeasurement is made possible. Therefore, the third method of the presentinvention is useful for a measuring method using the above reactionsystem.

[0058] The fourth method of the present invention is not particularlylimited if it is a method using a reaction system including theformation reaction of a detectable substance insoluble in a reactionsolvent or an insoluble by-product.

[0059] The measuring method of the present invention is used in a methodof detecting and determining an analyte, preferably a biologicalcomponent in the body fluid such as urine and blood, a trace amount of asubstance existent in food, medicine, or natural environment, anindustrial chemical substance, or a trace amount of a substancecontained in waste, from a sample containing the same.

[0060] The present invention provides a analytical testing piece formeasuring an analyte by measuring a detectable substance by using areaction system including a formation reaction of the detectablesubstance based on a chemical reaction of the analyte contained in asample, wherein the testing piece comprises at least one test portionhaving a detection portion for detecting the detectable substance andcontains a layered inorganic compound at least in the test portion. Thetesting piece will be referred to as “testing piece of the presentinvention” hereinafter.

[0061] The testing piece of the present invention may comprise at leastone test portion composed of two or more layers including a detectionlayer for detecting a detectable substance as the detection portion andcontain the layered inorganic compound at least in the detection layer.The testing piece of the present invention may be one in which the testportion further include a diffusion layer for diffusing a sample so thatthe sample passes through the diffusion layer to be diffused and reachesthe detection layer. The testing piece of the present invention maycomprise at least one test portion having a detection area for detectingthe detectable substance as the detection portion and contain thelayered inorganic compound at least in the detection area. The testingpiece of the present invention may be one in which the test portion hasa diffusion area for diffusing the sample so that the sample passesthrough the diffusion area to be diffused and reaches the detectionarea. Further, the testing piece of the present invention may be one inwhich the detection area composed of at least two layers including adetection layer for detecting the detectable substance. Moreover, thetesting piece of the present invention may be one in which the testportion has a reaction portion where the analyte contained in the samplereacts with a reagent react, and the detectable substance is formed inthe reaction portion. Further, the testing piece of the presentinvention may be one in which the detection portion is provided at alocation which the sample reaches after the sample is diffused andpasses through the reaction portion. Still further, the testing piece ofthe present invention may be one in which the detectable substance isformed by a reaction between the analyte contained in the sample and areagent in the detection portion.

[0062] In the testing piece of the present invention, it is consideredthat a dyestuff or the like formed by a reaction between an analyte anda reagent is adsorbed to a layered inorganic compound by including thelayered inorganic compound in the test portion with the result that thediffusion or elution of the dyestuff or the like by a sample solution ora reaction solution can be suppressed, and highly sensitive and highlyaccurate analysis is made possible.

[0063] The testing piece of the present invention is applied to a methodfor analyzing a component contained in a liquid using a solid phase,particularly analysis of glucose, bilirubin or the like contained inurine. In the analysis of the component contained in the liquid, adyestuff or the like formed by a reaction between an analyte and areagent may readily dissolve in a sample, diffuse and elute. Therefore,the testing piece of the present invention is effective.

[0064] The reagent is not particularly limited if it causes a detectablereaction with an analyte. It is preferably a reagent capable of forminga detectable substance such as a dyestuff compound, an oxidized/reducedform of an electron carrier or a complex compound of an ionophore and anion by reacting with the analyte. The formation reaction of a dyestuffcompound may be any reaction if it forms an optical detectablesubstance. It may be a reaction which causes not only color developmentbut also color change, fluorescence and emission. When the formeddyestuff compound or the like is water-soluble, it is often diffused andeluted by a sample solution, a reaction solution or the like. Therefore,the testing piece of the present invention is particularly preferablyapplied to a method using a reagent for forming such a water-solubledyestuff compound.

BRIEF DESCRIPTION OF THE DRAWINGS

[0065]FIG. 1 shows absorption spectra measured in Example 1.

[0066]FIG. 2 shows the calibration curves of hydrogen peroxide obtainedin Examples 2 and 3.

[0067]FIG. 3 shows logarithmic representation in both the axis ofordinate and the axis of abscissa of the calibration curves of FIG. 2.

[0068]FIG. 4 shows time-cource of absorbance after the addition ofhydrogen peroxide in Example 4.

[0069]FIG. 5 shows absorption spectra measured in Example 5.

[0070]FIG. 6 shows the calibration curves of the concentration ofascorbic acid obtained in Example 6.

[0071]FIG. 7 shows absorption spectra (smectite-added system) measuredin Example 7.

[0072]FIG. 8 shows absorption spectral(smectite-non-added system)measured in Example 7.

[0073]FIG. 9 shows absorption spectra (smectite-added system andnon-added system) at a sodium nitrite concentration of 33 mmol/lmeasured in Example 7.

[0074]FIG. 10 shows the calibration curves of the concentration ofsodium nitrite obtained in Example 8.

[0075]FIG. 11 shows time-cource of absorbance in experiments showing asmectite addition effect in a POD color developing system conducted inExample 9.

[0076]FIG. 12 shows time-cource of absorbance in experiments showing asmectite addition effect in a POD color developing system containingascorbic acid conducted in Example 10.

[0077]FIG. 13 is an enlarged view of a section for 0 to 60 seconds ofFIG. 12.

[0078]FIG. 14 shows time-cource of absorbance measured in Example 11.

[0079]FIG. 15 shows time-cource of absorbance in a smectite-non-addedsystem measured in Example 12.

[0080]FIG. 16 shows time-cource of absorbance in a smectite-added systemmeasured in Example 12.

[0081]FIG. 17 shows time-cource of absorbance in a smectite-added systemand a smectite-non-added system when the concentration of sodium nitriteis 25.0 mmol/l measured in Example 12.

[0082]FIG. 18 schematically shows the diffusion state of a dyestuff onsmectite-impregnated filter paper in Example 13.

[0083]FIG. 19 schematically shows the diffusion state of a dyestuff onuntreated filter paper in Example 13.

[0084]FIG. 20 schematically shows a reaction cell in Example 15.

[0085]FIG. 21 schematically shows a testing piece in Example 16.

[0086]FIG. 22 schematically shows a testing piece in Example 17.

[0087] In these figures, reference numeral 1 is a case where smectite isadded, 2 is a case where smectite is not added and 3 is a case wheresmectite is added and hydrogen peroxide is not added. 4 is a case wherethe concentration of nitrous acid is 33 μmol/l, 5 is a case where theconcentration of nitrous acid is 16 μmol/l, 6 is a case where theconcentration of nitrous acid is 8 μmol/l, and 7 is a case where theconcentration of nitrous acid is 0 μmol/l. 8 is sample No. 1, 9 issample No. 2, 10 is sample No. 3, 11 is sample No. 4, and 12 is sampleNo. 5. 13 is a case where the concentration of sodium nitrite is 50.0μmol/l, 14 is a case where the concentration of sodium nitrite is 25.0μmol/l, 15 is a case where the concentration of sodium nitrite is 12.5μmol/l, 16 is a case where the concentration of sodium nitrite is 6.3μmol/l, and 17 is a case where the concentration of sodium nitrite is1.6 μmol/l. 18 is a spot of a dyestuff, 19 is a color developingsolution from which a dyestuff is removed, 20 is glass, 21 is a coatingfilm, and 22 is PET. 23 is filter paper impregnated with a reagent(detection layer), 24 is adhesive double-coated tape (adhesive layer),25 is filter paper impregnated with a dispersion of a layered inorganiccompound, 26 is filter paper impregnated with a reagent and 27 is filterpaper. 28 is a sample suction area, 29 is a diffusion area, 30 is areaction area, 31 is an area for controlling reaction time, 32 is aholding area and 33 is an area for absorbing excess of a sample.

BEST MODE FOR CARRYING OUT THE INVENTION

[0088] I. Measuring Method of the Present Invention

[0089] The measuring method of the present invention is a method formeasuring an analyte by measuring a detectable substance by using areaction system including the formation reaction of the detectablesubstance based on the chemical reaction of the analyte contained in asample.

[0090] 1. Detectable Substance

[0091] The reaction system used in the present invention includes theformation reaction of the detectable substance as shown below.

[0092] The detectable substance is not particularly limited if it can beadsorbed by the layered inorganic compound according to the presentinvention. Examples of the detectable substance which can be adsorbed bythe layered inorganic compound include amines such as amine andpolyamine; imines such as imine and polyimine; polyenes; aromaticcompounds such as aniline derivatives, benzoquinone derivatives andaromatic condensed ring compounds; heterocyclic compounds such asxantene, azine and thiazine; complexes between ions and cyclic ligandssuch as crown ether and valinomycin; and the like. These substances maycontain a quaternary nitrogen atom, phenolic hydroxyl group, sulfonicacid group or carboxyl group in the molecule.

[0093] The substance which can be adsorbed by the layered inorganiccompound is detailed in, for example, Chapter XI “Interaction of Claysand Organic Compounds” of “An Introduction to Clay Colloid Chemistry,Second Edition” written by H. Van Olphen (Krieger Publishment, Malabar).Japanese Patent Publication No. 50-8462(1975) (Tadayoshi Kato)introduces many adsorbable compounds. They include substances which canbe detected by an optical method and an electrochemical method. (1)Substances which can be Detected by an Optical Method

[0094] Substances which can be detected by an optical method includedyestuffs. The dyestuffs include fluorescent dyestuffs, luminescentsubstances and the like. The formation reaction of the dyestuff may bethe formation reaction of an optically detectable substance, forexample, a reaction causing color development, color change,fluorescence, light emission or the like.

[0095] Preferred dyestuffs include dyestuff compounds, fluorescentsubstances and luminescent substances formed from dyestuff precursors byvarious coloration reactions such as an oxidation-reduction reaction, anacid-base reaction and a condensation reaction, dyestuff complexes andfluorescent complexes formed by coordinate bonding or ion bonding, andthe like.

[0096] Preferred dyestuffs formed by an oxidation-reduction reaction arecompounds having a conjugate system such as an aromatic ring, such asdyestuffs formed by the oxidation condensation of a coupler such as4-amino-1,2-dihydro-1,5-dimethyl-2-phenyl-3H-pyrazol-3-one(4-aminoantipyrin:to be abbreviated as 4-AA hereinafter) and a hydrogen donor (Trinder'sreagent such as N-ethyl-N-(3-sulfopropyl)-3,5-dimethylaniline);color-producing dyestuffs by oxidation such as o-tolidine and benzidines(such as 3,3′,5,5′-tetramethyl-benzidine); dyestuffs formed by theoxidation of leuco substances such as2,6-dichloro-4-[(4-hydroxyphenyl)imino]-2,5-cyclohexadien-1-one;fluorescent substances formed by the oxidation of 4-hydroxyphenylacetate; luminescent substances such as chemiluminescent substances andexciting substances thereof; formazans which are reducing dyestuffs oftetrazolium salts; dyestuffs formed by the reduction of1,1′-dimethyl-4,4′-bipyridinium salts and the like; and the like.

[0097] The hydrogen donor is a compound such as phenol which forms aquinone dyestuff when it is condensed with4-amino-1,2-dihydro-1,5-dimethyl-2-phenyl-3H-pyrazol-3-one(4-AA) or3-methyl-2-benzothiazoline hydrazone by the function of peroxidase inthe presence of hydrogen peroxide. Examples of the hydrogen donorinclude dichlorophenol, o-methoxyphenol, 1,2,3-trihydroxybenzene,dimethylaniline, N-ethyl-N-sulfopropyl-m-anisidine,N-ethyl-N-sulfopropylaniline,N-ethyl-N-(3-sulfopropyl)-3,5-dimethoxyaniline,N-ethyl-N-(3-sulfopropyl)-3,5-dimethylaniline,N-ethyl-N-sulfopropyl-m-toluidine,N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-anisidine,N-ethyl-N-(2-hydroxy-3-sulfopropyl)aniline,N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline,N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline,N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline,N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine,N-(3-sulfopropyl)aniline and the like.

[0098] In a method using a reaction system which forms a quinonedyestuff by a reaction between the above 4-AA and a hydrogen donor inthe presence of hydrogen peroxide, an analyte is measured by measuringhydrogen peroxide indirectly by measuring the formed quinone dyestuffwith an absorptiometer.

[0099] o-Tolidine and benzidines include o-tolidine, dianisidine,3,3′-diaminobenzidine, 3,3′,5,5′-tetra-methylbenzidine,N-(3-sulfopropyl)-3,3′,5,5′-tetra-methylbenzidine and the like.

[0100] The leuco substance is an achromatic dyestuff precursor whichbecomes a dyestuff and develops color when it is oxidized. Examples ofthe dyestuff obtained by oxidizing the leuco substance include2,6-dichloro-4-[(4-hydroxyphenyl)imino]-2,5-cyclohexadien-1-one,2,6-dichloro-4-[(3-chloro-4-hydroxyphenyl)imino]-2,5-cyclohexadien-1-one,7-(diethylamino)-3-imino-8-methyl-3H-phenoxazine salt,3-(diethylamino)-7-amino-5-phenylphenazinium salt,3,7-bis(dimethylamino)-phenothiazin-5-ium salt,1-hydroxy-5-methylphenazinium salt, and7-hydroxy-3H-phenoxazin-3-one-10-oxide. Examples of the leuco substanceinclude 4,4′-benzylidenebis(N,N-dimethylaniline),4,4′-bis[N-ethyl-N-(3-sulfopropylamino)-2,6-dimethylphenyl]methane,1-(ethylaminothiocarbonyl)-2-(3,5-dimethoxy-4-hydroxyphenyl)-4,5-bis(4-diethylaminophenyl)imidazole,4,4′-bis(dimethylamino)diphenylamine,N-(carboxymethylaminocarbonyl)-4,4′-bis(dimethylamino)-diphenylaminesalt,10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)phenothiazine saltand the like.

[0101] Other examples of the dyestuff precursor which develops colorwhen it is oxidized include 4-methoxyphenol, 4-ethoxyphenol,2-ethoxyphenol, 1-(2-hydroxy-5-methoxyphenyl)ethanone,2-hydroxy-5-methoxybenzoic acid, 2-hydroxy-5-methoxybenzaldehyde,2-hydroxy-5-methoxymethoxybenzoic acid, 4-methoxy-2-nitrophenol,2-chloro-4-methoxyphenol, 4-hydroxy-3-methoxybenzaldehyde,4-hydroxy-3-methoxybenzoic acid and the like.

[0102] 3-(4-Hydroxyphenyl)-2-propenoic acid, 2-hydroxyphenylacetic acid,3-hydroxyphenylacetic acid, 4-hydroxyphenylacetic acid, 3-hydroxybenzoicacid, 4-hydroxybenzoic acid, 2-aminobenzoic acid, 3-aminobenzoic acid,4-aminobenzoic acid, 3,4-diaminobenzoic acid, 3,5-diaminobenzoic acid,4-amino-2-chlorobenzoic acid, 4-amino-3-methylbenzoic acid,4-amino-3-methoxybenzoic acid, 4-aminophthalic acid and the like arefurther included in the above examples.

[0103] 2,4-Diamino-6-hydroxypyrimidine, 4,5-diamino-6-hydroxypyrimidine,4-amino-2,6-dihydroxypyrimidine, 6-hydroxy-2,4,5-triaminopyrimidine,4,5-diamino-2,6-dihydroxypyrimidine,4-amino-6-hydroxy-2-methylpyrimidine, 4-amino-6-hydroxypyrimidine,4-amino-6-hydroxy-2-methoxypyrimidine and the like are also included.

[0104] Fluorescent substances may be formed by oxidizing4-hydroxyphenylacetic acid and the like. Examples thereof includefluorescent substances formed by oxidizing 4-hydroxyphenylacetic acid,(4-hydroxy-3-methoxypehnyl)acetic acid, 3-(4-hydroxyphenyl)propionicacid, 4-hydroxy-(2-aminoethyl)-phenol,4-hydroxy-N,N,N-trimethylbenzenemetaminium,α-amino-p-hydroxyhydrocinnamic acid, 4-hydroxyphenethylamine,N-(4-hydroxyphenyl)acetoanilide, 2,7-dichlorofluorescein diacetate andthe like.

[0105] Luminescent substances such as chemiluminescent substancesinclude firefly luciferin, Cypridina luciferin, aequorin, lucigeninderivatives, luminol derivatives, acridinium esters, peroxalic acidesters and the like.

[0106] For example, in the above method using a reaction system in whichthe benzidine or leuco substance is oxidized in the presence of hydrogenperoxide and develops color, an analyte is measured by measuringhydrogen peroxide indirectly by measuring the formed dyestuff with anabsorptiometer.

[0107] In the above method using a reaction system in which afluorescent substance or a luminescent substance is formed, an analyteis measured by measuring hydrogen peroxide indirectly with afluorophotometer or a luminophotometer.

[0108] In the oxidation reaction which forms a dyestuff, an oxidizingagent used for the oxidation reaction is not limited to hydrogenperoxide but various known oxidizing agents may be used. An oxidizingenzyme such as peroxidase may be added. Prior to the oxidation reactionwhich forms a dyestuff, a reaction for forming the oxidizing agent maytake place.

[0109] The tetrazolium salts include 2,3,5-triphenyl-tetrazolium salt,2,5-diphenyl-3-(1-naphthyl)-2H-tetrazolium salt,3,3′-(3,3′-dimethoxy-4,4′-biphenylene)-bis[2-(p-nitrophenyl)-5-phenyl-2H-tetrazolium]salt,3,3′-[3,3′-dimethoxy-(1,1′-biphenyl)-4,4′-diyl]-bis(2,5-diphenyl-2H-tetrazolium)salt,2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazolium salt,2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazoliumsalt,2-(4-iodophenyl)-3-(2,4-dinitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazoliumsalt,3,3′-(1,1′-biphenyl-4,4′-diyl)-bis(2,5-diphenyl-2H-tetrazolium)salt,3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium salt and thelike.

[0110] The dyestuffs formed by reduction include reduced forms of1,1′-dimethyl-4,4′-bipyridinium salt, 1,1′-dibenzyl-4,4′-bipyridiniumsalt and the like. Fluorescent substances may be formed by reducing7-hydroxy-3H-phenoxazin-3-one-10-oxide and the like. Examples thereofinclude fluorescent substances formed by reducing7-hydroxy-3H-phenoxazin-3-one-10-oxide,5-cyano-2,3-bis(4-methylphenyl)-2H-tetrazolium salt,2,3-bis(4-cyanophenyl)-5-cyano-2H-tetrazolium salt and the like.

[0111] For example, in the above method using a reaction system in whichthe tetrazolium salt or the leuco substance is reduced in the presenceof a reducing agent and develops color, an analyte is measured bymeasuring the reducing agent indirectly by measuring the formed dyestuffwith a colorimenter or fluorophotometer. Prior to the reduction reactionwhich forms a dyestuff, a reaction for forming a reducing agent may takeplace.

[0112] In the above reduction reaction which forms a dyestuff,nicotinamide adenine dinucleotide or nicotineamide adenine dinucleotidephosphate is preferably used as the reducing agent used for thereduction reaction. However, the present invention is not limited tothis but various known reducing agents may be used.

[0113] Dyestuffs formed by an acid-base reaction include compounds whichdevelop color or change their colors by pH variations, such asBromocresol Green. The compounds include sulfone phthalein dyestuffssuch as Bromophenol Blue, Phenol Red, Bromopyrogallol Red and PyrogallolRed; triphenylmethane dyestuffs such as Malachite Green and rosolicacid; quinoline dyestuffs such as Quinaldine Red,N-(p-hydroxyphenyl)-2,6-dichloro-p-benzoquinoneimine; oxazone dyestuffssuch as 7-hydroxy-3H-phenoxazin-3-one 10-oxide; coumarin dyestuffs suchas 6,7-dihydroxy-4-methylcoumarin; conductive polymer compounds such asaniline oligomer, and the like, in addition to Bromocresol Green.

[0114] For example, in the method using a reaction system in which acompound which develops color or changes in color by pH variationsdevelops color or changes its color by an acid or a base, an analyte ismeasured by measuring an acid or base indirectly by measuring the formeddyestuff with an absorptiometer. In the method using a reaction systemin which a compound which develops color or changes in color by pHvariations develops color or changes its color by hydrogen ions, ananalyte is measured by measuring the concentration of hydrogen ions bymeasuring the formed dyestuff with an absorptiometer.

[0115] Other dyestuffs which are formed by various reactions known as acoloration reaction and the like include azo dyestuffs formed by thecoupling of a diazonium salt such as2-methoxy-4-morpholinobenzenediazonium salt; dyestuffs formed by variousknown coloration reactions such as a reaction between aldehyde and2,3-dimethyl-2,3-bis(hydroxyamino)butane; fluorescent substances formedby various known reactions such as a reaction between histamine ando-phthalaldehyde; and dyestuffs and fluorescent substances formed by thereaction of an enzyme substrate such as 4-methylumbelliferyl phosphateby an enzyme.

[0116] The azo dyestuffs formed by the coupling of a diazonium saltinclude azo dyestuff formed by coupling between indoxyl and2-methoxy-4-morpholinobenzenediazonium salt, azo dyestuff formed bycoupling between urobilinogen and 3,3′-dimethoxybiphenyl-4,4′-diazoniumsalt, azo dyestuff formed by a reaction between 4-aminobenzenearsonicacid and N-1-naphthylethylene diamine in the presence of a nitrite, azodyestuff formed by a reaction between 2,4-dichloroaniline andN,N-diethyl-N′-1-naphthylnaphthylethylene diamine oxalate in thepresence of a nitrite, and the like.

[0117] In the above method using a reaction system in which an azodyestuff is formed, an analyte (indoxyl, urobilinogen and nitrite in theabove examples) as the starting substance of a reaction is measured bymeasuring the formed dyestuff with an absorptiometer or the like. Theformation reaction of an azo dyestuff is not limited to the aboveexamples but various known formation reactions of azo dyestuffs aresuitably applied.

[0118] Dyestuffs formed by various known coloration reactions includedyestuffs formed by the following known coloration reactions, but thepresent invention is not limited to these as a matter of course. Thecoloration reactions include a reaction between hydrogen peroxide and1,4-diaminobenzene for detecting aldehyde, the reaction of2,3-dimethyl-2,3-bis(hydroxyamino)butane for detecting aldehyde, areaction between 3-methyl-2-benzothiazolinonehydrazone and an oxidizingagent for detecting aldehyde, a reaction between 10H-phenothiazine andbromine for detecting secondary amine, the reaction of2,2′-dithiodipyridine for detecting thiol and the like.

[0119] In the above method using a known coloration reaction, an analyte(aldehyde, secondary amine and thiol in the above examples) as thestarting substance of a reaction is measured by measuring the formeddyestuff with an absorptiometer or the like. The known colorationreaction usable is not limited to the above examples as a matter ofcourse.

[0120] Fluorescent substances formed by various known reactions includefluorescent substances formed by known detection reactions which arecarried out using the following reagents. However, the present inventionis not limited to these as a matter of course. The reagents used in thedetection reactions which form fluorescent substances include2-hydroxy-1,2-diphenylethanone for detecting a guanidino compound,o-phthalaldehyde for detecting histamine, o-phthalaldehyde for detectingspermidine, 1,2-diamino-4,5-dimethoxybenzene for detecting α-keto acidand the like.

[0121] In the above method using a known detection reaction, an analyte(guanidino compound, histamine, spermidine and α-keto acid in the aboveexamples) as the starting substance of a reaction is measured bymeasuring the formed fluorescent substances with a fluorophotometer orthe like. The known detection reaction usable is not limited to theabove examples as a matter of course.

[0122] Enzyme substrates which form dyestuffs and fluorescent substanceswhen they react in the presence of enzymes includeN-tosyl-L-phenylalanine-2-amidoacridone as the substrate ofchymotrypsin, L-alanine-2-amidoacridone as the substrate ofaminopeptidase, 7-acetoxy-N-methylquinolinium salt for measuringesterase, 7-acetoxy-3H-phenoxazin-3-one as the substrate of esterase,4-methylumbelliferyl phosphate as the substrate of phosphatase,5,10,15,20-tetrakis(4-phosphonooxyphenyl)porphine as the substrate ofphosphatase and the like. The present invention is not limited to theseas a matter of course.

[0123] For example, in the above method using a reaction in which anenzyme substrate is decomposed by an enzyme, an analyte is measured bymeasuring an enzyme indirectly by measuring the formed dyestuff or thefluorescent substance with an absorptiometer or a fluorophotometer. Theenzyme or the enzyme substrate may be chemically bonded to an antibodyor a fragment thereof, for example.

[0124] Dyestuff complexes and fluorescent complexes formed by coordinatebonding and ion bonding include compounds which develop color or changetheir colors, such as dyestuffs and fluorescent substances produced byforming complexes by ion bonding or coordinate bonding between a metalion or anion and a compound such as a ligand. The compounds whichdevelop color or change their colors and form complexes with a metal ioninclude compounds known as metal indicators and chromoionphores andcompounds which are colored by forming complexes with a coloredtransition metal ion. Specifically, the compounds include ethylenediamine tetraacetic acid, 2,2-bipyridine,1-hydroxy-2-(2-hydroxyphenylazo)benzene, dibenzo-18-crown-6,dicyclohexyl-18-crown-6, cyclic polyamines, calix[4]arene,3-[N,N-bis-(carboxymethyl)aminomethyl]-1,2-dihydroxyanthraquinone,5′,5″-dibromopyrogallolsulfone phthalein,2-hydroxy-1-(1-hydroxy-2-naphthylazo)-6-nitro-4-naphthalene sulfonate,2,6-dichloro-4′-hydroxy-3,3″-dimethyl-fuchsone-5′,5″-dicarboxylate,3,3′-bis[N,N-bis-(carboxymethyl)aminomethyl]fluorescein,8-[N,N-bis(carboxymethyl)aminomethyl]-4-methylumbelliferone,2,7-bis(2-arsonophenylazo)-1,8-dihydroxy-3,6-naphthalene disulfonicacid, 5-chloro-2-hydroxy-3-(2,4-dihydroxyphenylazo)benzenesulfonic acid,5-[(hexahydro-2,4,6-trioxo-5-pyrimidinyl)imino]-2,4,6-(1H,3H,5H)-pyrimidinetrionesalt,2-(5-bromo-2-pyridylazo)-5-[N-propyl-N-(3-sulfopropyl)amino]anilinesalt, 1,8-dihydroxy-2-(2-pyridylazo)-3,6-naphthalene disulfonate,2-nitroso-5-[N-propyl-N-(3-sulfopropyl)amino]phenol and the like.

[0125] Compounds which form colored complexes with monovalent cationsspecifically include tetrakis[3,5-bis-(trifluoromethyl)phenyl]boratesalt, tetraphenylphosphonium salt and the like.

[0126] Compounds which form fluorescent complexes with calcium ionsspecifically include1-[2-amino-5-(2,7-dichloro-6-hydroxy-3-oxy-9-xanthenyl)phenoxy]-2-(2-amino-5-methylphenoxy)ethane-N,N,N′,N′-tetraacetate,1-[2-amino-5-(2,7-dichloro-6-hydroxy-3-oxy-9-xanthenyl)phenoxy]-2-(2-amino-5-methylphenoxy)ethane-N,N,N′,N′-tetraaceticacid-pentaacetoxymethyl ester,1-[6-amino-2-(5-carboxy-2-oxazoyl)-5-benzofuranyloxy]-2-(2-amino-5-methylphenoxy)ethane-N,N,N′,N′-tetraacetate,1-[6-amino-2-(5-carboxy-2-oxazoyl)-5-benzofuranyloxy]-2-(2-amino-5-methylphenoxy)ethane-N,N,N′,N′-tetraaceticacid-pentaacetoxymethyl ester,1-[2-amino-5-(6-carboxy-2-indolyl)phenoxy]-2-(2-amino-5-methylphenoxy)ethane-N,N,N′,N′-tetraacetate,1-[2-amino-5-(6-carboxy-2-indolyl)phenoxy]-2-(2-amino-5-methylphenoxy)ethane-N,N,N′,N′-tetraaceticacid-pentaacetoxymethyl ester,8-amino-2-[(2-amino-5-methylphenoxy)methyl]-6-methoxyquinoline-N,N,N′,N′-tetraacetate,8-amino-2-[(2-amino-5-methylphenoxy)methyl]-6-methoxyquinoline-N,N,N′,N′-tetraaceticacid-pentaacetoxymethyl ester,3,3′-bis[N,N-bis(carboxymethyl)aminomethyl]fluorescein,8-[N,N-bis(carboxymethyl)aminomethyl]-4-methyl-umbelliferone and thelike.

[0127] Further, a tetraphenylarsonium salt which forms a colored complexwith an anion, N-ethoxycarbonylmethyl-6-methoxyquinolinium bromide whosefluorescent intensity is reduced when it forms a complex with a chlorideion, 8-hydroxy-1-(salicylideneamino)-3,6-naphthalene disulfonate whichforms a complex with boron and the like are also included.

[0128] In the above method using the formation reaction of a complex, ananalyte (ions in most cases) is measured by measuring the amount of adyestuff or a fluorescent substance formed by an ion and a ligand withan absorptiometer or a fluorophotometer.

[0129] (2) Electrochemically Detectable Substances

[0130] A description is subsequently given of electrochemicallydetectable substances.

[0131] Substances which can be detected by electrochemical methodsinclude electron carriers (mediators) and complexes between ionophoresand ions.

[0132] The electron carrier is a chemical substance whichoxidizes/reduces an analyte with an enzyme or the like andreceives/gives electrons directly from/to the analyte at the same time.The analyte can be measured from electrochemical response when theoxidized/reduced form of the electron carrier is oxidized/reduced by anelectrode. The electron carrier does not need to receive/give electronsdirectly from/to the analyte and may be a chemical substance whichoxidizes/reduces the analyte with an enzyme or the like andreceives/gives electrons indirectly from/to the analyte at the sametime. The analyte is measured from electrochemical response when theoxidized/reduced form of the electron carrier having quantitativerelationship with the analyte is reduced/oxidized by an electrode.

[0133] The electron carrier is preferably oxidized/reduced by apotential within a range which can be measured by the used electrode(generally −1.2 V to +1.0 V in the case of a carbon electrode). Theelectron carrier is exemplified by 1,1′-dimethyl-4,4′-bipyridinium salt,1,1′-dibenzyl-4,4′-bipyridinium salt, 1,4-diaminobenzene,2-methyl-1,4-naphthoquinone, N-methylphenazinium salt,1-hydroxy-5-methylphenazinium salt, 1-methoxy-5-methylphenazinium salt,9-dimethylaminobenzo-α-phenoxazin-7-ium salt, ferrocene derivative,hexacyano iron(II) salt, 7-hydroxy-3H-phenoxazin-3-one-10-oxide,3,7-diamino-5-phenylphenazinium salt,3-(diethylamino)-7-amino-5-phenylphenazinium salt, 1,4-benzenediol,1,4-dihydroxy-2,3,5-trimethylbenzene,N,N,N′,N′-tetramethyl-1,4-benzenediamine, A2,2′-bi-1,3-dithiol,2,6-dimethyl-benzoquinone, 2,5-dimethylbenzoquinone,2,3,5,6-tetramethyl-2,5-cyclohexadiene-1,4-dione,2,6-dichloro-4-[(4-hydroxyphenyl)imino]-2,5-cyclohexadien-1-one,2,6-dichloro-4-[(3-chloro-4-hydroxyphenyl)imino]-2,5-cyclohexadien-1-one,7-(diethylamino)-3-imino-8-methyl-3H-phenoxazine salt,3,7-bis(dimethylamino)-phenothiazine-5-ium salt and the like.

[0134] Detectable substances in this case are oxidized/reduced forms ofthe above electron carriers, and the formation reaction of thesedetectable substances is the oxidation/reduction reaction of theelectron carriers. As described above, an analyte can be measured bymeasuring electrochemical response such as an oxidation/reductioncurrent when the oxidized/reduced form of an electron carrier existenthaving quantitative relationship with the analyte is reduced/oxidized byan electrode. For example, the analyte can be indirectly measured fromthe result of electrochemical response measured when the electroncarrier is oxidized/reduced on an electrode as an electrondonor/receptor like ascorbic acid or hydrogen peroxide.

[0135] The ionophore is a compound such as a ligand which iscoordinately bonded or ion bonded selectively to a specific ion as ananalyte to become a complex and it is particularly well known that theionophore is used in a liquid film electrode.

[0136] Examples of the ionophore which forms a complex with a cationinclude tetrakis[3,5-bis(trifluoromethyl)phenyl)borate salt,tetraphenylphosphonium salt, valinomycin,cyclo(N′,N′-dioctyl-D-asparaginyl-L-prolyl-L-alanyl)₂,bis(benzo-15-crown-5), bis[(benzo-15-crown-5)-4-methyl]pimelate,bis(12-crown-4), bis[(12-crown-4)methyl]-2-dodecyl-2-methylmalonate,14-crown-4, dodecyl-methyl-14-crown-4,6,6-dibenzyl-1,4,8,11-tetraoxacyclotetradecane, dibenzo-18-crown-6,dicyclohexyl-18-crown-6,4,16-di-N-octadecylcarbamoyl-3-oxabutyryl-1,7,10,13,19-pentaoxa-4,16-diazacyclohenicosane and the like.

[0137] Examples of the ionophore which forms a complex with an anioninclude tetraphenylarsonium salt, 6-methoxy-N-(3-sulfopropyl)quinoliniumsalt and the like.

[0138] The liquid film electrode is used in a method of measuring aspecific ion as an analyte, by measuring a membrane potential producedwhen a porous polymer layer is formed on the surface of an electrode,and an ionophore is infiltrated into the polymer layer, bonded to thespecific ion contained in a sample and moved in the polymer layer toselectively separate the specific ion. It is a matter of course that theuse of the ionophore in the electrochemical detection method is notlimited to this liquid film electrode.

[0139] A specific ion as an analyte can be measured by measuring amembrane potential produced when the specific ion is bonded to anionophore in a bulk solution and the ion is selectively separated in anelectrode having a polymer layer in which unbonded ions cannot move andonly a complex produced by bonding can move.

[0140] In this case, the detectable substance is a complex between theionophore and the specific ion, and the formation reaction of thedetectable substance is a complex formation reaction between theionophore and the specific ion by coordinate bonding or ion bonding. Ananalyte is measured by electrochemically measuring a membrane potentialproduced according to the concentration of a specific ion which is theanalyte as described above.

[0141] 2. Measuring Method which the Present Invention is Applied to

[0142] The measuring method of the present invention is preferablyapplied to the method using a reaction system which forms such adetectable substance, more preferably to the following methods.

[0143] (a) A method using a reaction system including the formationreaction of hydrogen peroxide or an oxidization reaction using hydrogenperoxide as an oxidizing agent, specifically a method for measuring adyestuff compound produced by forming hydrogen peroxide from an analytethrough an oxidase reaction system and carrying out anoxidation-reduction reaction between the hydrogen peroxide and areactive color producer (dyestuff precursor) in the presence ofperoxidase.

[0144] (b) A method using a reaction system including the formationreaction of nicotinamide adenine dinucleotide (NADH) or nicotinamideadenine dinucleotide phosphate (NADPH) or a reaction using NADH or NADPHas a reducing agent, specifically a method for measuring a dyestuffcompound produced by forming NADH or NADPH from an analyte through adehydrogenase reaction system and reducing it by making it act on adyestuff precursor in the presence of an electron carrier.

[0145] (c) A method for measuring a nitrite, diazqnium salt or couplingcompound by producing a diazonium salt by allowing a nitrite to reactwith an aromatic primary amine under an acidic condition to produce adiazonium salt, allowing the produced diazonium salt to react with areagent to be coupled to produce an azo dyestuff and measuring theproduced azo dyestuff.

[0146] (d) A method for measuring a substance labeled with a fluorescentenzyme substrate or an alkali phosphatase by measuring fluorescencegenerated by producing a fluorescent substance by isolating a phosphatefrom a fluorescent enzyme substrate such as 4-methyl-umbelliferonehaving a phosphoric acid ester by the function of alkali phosphatase andirradiating the fluorescent substance with excitation light.

[0147] (e) A method for measuring an oxidase/reductase and a substancelabeled with an oxidase/reductase by measuring a current response when amediator such as 1,4-diaminobenzene is oxidized/reduced by theoxidase/reductase and the produced oxidized/reduced form of the mediatoris reduced/oxidized by an electrode reaction.

[0148] The detectable substance may be the analyte. As an examplethereof, an analyte is measured by measuring electrochemical responsewhen glucose dissolved in water is oxidized on the surface of anelectrode.

[0149] The fourth measuring method of the present invention may beparticularly a measuring method using a reaction system which forms adetectable substance in a solvent and including the formation reactionof a substance insoluble in the solvent, preferably to a measuringmethod in which an insoluble detectable substance is formed,particularly preferably to a measuring method in which the detectablesubstance is an optically detectable substance.

[0150] The solvent is not particularly limited and conventionally knownsolvents can be used. Examples of the solvent include water such asdistilled water, alcohols such as ethanol, ketones such as acetone,ethers such as diethyl ether, esters such as ethyl acetate, halogenatedhydrocarbons such as chloroform, aromatic hydrocarbons such as benzeneand toluene, and the like. A solvent suitable for an analyte and adetection reaction system thereof can be selected from these. Out ofthese, water is preferred. It is known in dry chemistry that a sampleliquid such as blood, saliva and urine containing an analyte can be usedas a reaction solvent.

[0151] The measuring method using the formation reaction of a substanceinsoluble in a reaction solvent in which the reaction is carried out isnot particularly limited. Examples thereof include a method using areaction which forms an optically detectable substance insoluble in areaction solvent, a method using a reaction which forms an opticallydetectable substance and a by-product insoluble in a solvent in whichthe reaction is carried out, and a method using a reaction which formsan electrochemically detectable substance and a by-product insoluble ina reaction solvent.

[0152] The measuring method using a reaction which forms an opticallydetectable substance insoluble in a reaction solvent is not particularlylimited but it may be one of the following detection reactions.

[0153] Oxidation reactions include a reaction for detecting hydrogenperoxide by the oxidation condensation of phenol and4-amino-1,2-dihydro-1,5-dimethyl-2-phenyl-3H-pyrazol-3-one in an aqueoussolution, a reaction for detecting hydrogen peroxide which formsN-methylacridone, a fluorescent substance insoluble in water, byoxidizing N-methylacridine-9-carboxylate in an aqueous solution, areaction for detecting hydrogen peroxide comprising a reaction foroxidizing an aqueous solution of10-(carboxymethyl-aminocarbonyl)-3,7-bis(dimethylamino)phenothiazinesalt in the presence of alkane sulfonate and causing it to developcolor, and the like.

[0154] Reduction reactions include a reaction for detecting a reducingsubstance which forms 7-hydroxy-3H-phenoxazin-3-one, a fluorescentsubstance insoluble in an acidic aqueous solution by reducing7-hydroxy-3H-phenoxazin-3-one-10-oxide, a reaction for detecting areducing substance which forms a formazan dyestuff insoluble in water byreducing3,3′-(3,3′-dimethoxy-4,4′-biphenylene)-bis[2-(p-nitrophenyl)-5-phenyl-2H-tetrazolium]salt,5-cyano-2,3-bis(4-methylphenyl)-2H-tetrazolium salt,2,3-bis(4-cyanophenyl)-5-cyano-2H-tetrazolium salt or the like, and thelike.

[0155] Other reactions include a reaction for detecting allyl sulfatasethrough a reaction between 4-methylumbelliferyl sulfate and arylsulfatase in a weak acidic aqueous solution, a reaction for detectingβ-glucosidase through a reaction between2-chloro-4-nitrophenyl-β-D-glucopyranoside and β-glucosidase in anaqueous solution, a reaction for detecting aldehyde through acondensation reaction between azobenzene-p-phenylhydrazinesulfonic acidand aldehyde, a reaction for detecting nitrous acid including diazocoupling between a diazonium salt and 2-naphthol, a reaction fordetecting a cobalt ion through a colored insoluble complex formationreaction between 1,3-diamino-4-(5-bromo-2-pyridylazo)benzene and acobalt ion in a neutral aqueous solution, and the like.

[0156] The measuring method using the formation reaction of an opticallydetectable substance, which includes the formation reaction of aby-product insoluble in a reaction solvent, or the measuring methodusing the formation reaction of an electrochemically detectablesubstance, which includes the formation reaction of a by-productinsoluble in a reaction solvent is rarely used because of inconveniencedescribed in the above section of the related art. Therefore, examplesof the above measuring methods are not enumerated herein. However, it isto be understood that the present invention is not limited by this.

[0157] It is a usually observed phenomenon that an insoluble substanceseparates out from a liquid phase immediately and is adsorbed to a solidphase nearby when it is formed in a solvent that does not dissolve it.If the solid phase does not exist nearby, the insoluble substance formsan assembly of molecules in a liquid phase, the assembly of moleculesfurther grows and settles as an aggregate or precipitate. The layeredinorganic compound of the present invention is a fine particle which isfine enough to be uniformly dispersed. Since insoluble detectablesubstances such as an insoluble detectable substance and a by-product inthe present invention are a single molecule or a very small assembly ofmolecules immediately after they are formed, the insoluble substancesare efficiently adsorbed to the layered inorganic compound and uniformlydispersed together.

[0158] The fourth measuring method of the present invention is ameasuring method which causes a layered inorganic compound having afunction to adsorb various kinds of substances and disperse uniformly toexist in a detection reaction system which forms an insoluble substance,thereby adsorbing the insoluble substance, and which overcomes suchinconvenience produced by the insoluble substance as described in thesection of the related art. In addition, the existence of the layeredinorganic compound does not interrupt the proceeding of a detectionreaction and detection. In the fourth measuring method of the presentinvention, the reaction system including the formation reaction of aninsoluble substance is particularly preferably the reduction reaction ofa tetrazolium salt.

[0159] Examples of the analyte which can be measured by the first tofourth measuring methods of the present invention include biologicalcomponents in the body fluid such as urine and blood, trace amounts ofsubstances existent in foods, medicines, or natural environment,industrial chemical substances, trace amounts of substances contained inwaste, and the like.

[0160] 3. Layered Inorganic Compound

[0161] The measuring method of the present invention is characterized inthat a layered inorganic compound is caused to exist in the reactionsystem including the formation reaction of the detectable substance. Adescription is subsequently given of the layered inorganic compound.

[0162] The layered inorganic compound used in the present invention isan inorganic compound having a crystal structure of sheet-structuredlayers piled up one upon another, each layer composed of a set ofpolyhedrons such as Si tetrahedrons or Al octahedrons arranged on thesame plane, as exemplified by layered clay minerals and hydrotalcite.

[0163] Clay minerals refer to aluminum silicate minerals which form themost part of clay (fine soil-like inorganic granular substance havingplasticity in a wet state). They are generally composed of minimumstructural units which are Si tetrahedrons having Si surrounded by 4 O's(oxygen atoms) and Al or Mg octahedrons having Al or Mg surrounded by 6OH groups or O's.

[0164] The structure of the layered clay mineral is one in which ahexagonal net sheet is formed with Si tetrahedrons sharing one plane andO's at the remaining vertices oriented in the same direction(tetrahedral sheet), or a sheet is formed with Al (or Mg) octahedronssharing ridgeangles (octahedral sheet), and these sheets are laminatedone upon another. Minerals having such a structure that a plurality of1:1 layers, each layer consisting of one tetrahedral sheet layer and oneoctahedral sheet layer, are laminated one upon another are called 1:1type minerals, minerals having such a structure that a plurality of 2:1layers, each layer consisting of one octahedral sheet layer and twotetrahedral sheet layers sandwiching the octahedral sheet layer, arelaminated one upon another are called 2:1 type minerals, and mineralshaving such a structure that another octahedral sheet layer insertedbetween the layers of 2:1 type are called 2:1:1 type minerals. Mineralswhich comprises Mg(OH)₂ octahedral sheets and have metal ions at alloctahedral positions are called trioctahedral type minerals and mineralswhich comprise Al(OH)₃ octahedral sheets and 1/3 of the octahedralpositions are empty holes are called dioctahedral type minerals. Thelayered inorganic compound used in the present invention is preferably a2:1 type mineral.

[0165] The layered inorganic compound used in the present inventionpreferably contains at least one element selected from lithium, sodium,potassium, magnesium, aluminum, silicon, oxygen, hydrogen, fluorine andcarbon. The layered inorganic compound may be a compound represented byany one of the following formulas. The compound represented by any oneof the following formulas may contain crystal water. These formulas arethe formulas of mineralogically and chemically pure compounds. Since theactual layered inorganic compound may contain impurities such as sodiumsilicate, the chemical formula of the layered inorganic compounddetermined by elemental analysis does not always agree with one of theseformulas as described in the document (D. W. Thompson, J. T.Butterworth, J. Colloid Interf. Sci., 151, 236-243 (1992)).

M_(x)Si₄(Al_(2-x)Mg_(x))O₁₂X₂  (1)

[0166] wherein, M is any one of H, Li, Na and K, X is any one of OH andF, and x is a positive number of less than 2.

M_(x)(Si_(4-x)Al_(x))Al₂O₁₀X₂  (2)

[0167] wherein, M is any one of H, Li, Na and K, and X is any one of OHand F, and x is a positive number of less than 4.

M_(x)Si₄(Mg_(3-x)Li_(x))O₁₀X₂  (3)

[0168] wherein, M is any one of H, Li, Na and K, X is any one of OH andF, and x is a positive number of less than 3.

M_(X)(Si_(4-x)Al_(x))Mg3O₁₀X₂  (4)

[0169] wherein, M is any one of H, Li, Na and K, X is any one of OH andF, and x is a positive number of less than 4.

MSi₄Mg_(2.5)O₁₀X₂  (5)

[0170] wherein, M is any one of Li, and Na, preferably Na, and X is anyone of OH and F, preferably F.

M₂Si₄Mg₂O₁₀X₂  (6)

[0171] wherein, M is any one of Li and Na, preferably Li, and X is anyone of OH and F, preferably F.

Mg₆Al₂(OH)₁₆X_(x)  (7)

[0172] wherein, X is any one of halogen, NO₃, SO₄, CO₃ and OH or ananion of an organic acid, preferably CO₃, and x is 2 when X is halogen,OH, NO₃ or a monovalent organic acid and 1 when X is SO₄, CO₃ or adivalent organic acid.

Na_(0.33)Si₄(Mg_(2.67)Li_(0.33))O₁₀X₂  (8)

[0173] wherein, X is either OH or F, preferably OH.

Na_(a-b)(Si_(4-a)Al_(a)) (Mg_(3-b)Al_(b))O₁₀X₂  (9)

[0174] wherein, X is either OH or F, preferably OH, a is a positivenumber of less than 4, and b is a positive number of less than 3,provided that a-b>0.

[0175] Examples of the layered inorganic compound used in the presentinvention include 1:1 type clay minerals such as kaolinite, halloysiteand serpentine; 2:1 type clay minerals such as talc, pyrophyllite,smectite, vermiculite (represented by the formula 2 out of the aboveformulas) and mica including fluorotetrasilicic mica (formula 5) andtaeniolite (formula 6); 2:1:1 type clay minerals such as chlorite; 2:1to 2:1:1 type intermediate minerals; para-amorphous clay minerals suchas imogolite; amorphous clay minerals such as allophane; hydrotalcite(formula 7); and the like.

[0176] According to ion species contained in the isomorphouslysubstituted tetrahedron and octahedron, the smectite is divided into adioctahedral type such as montmorillonite (formula 1), bentonite whichis a natural product containing 40 to 80% of montmorillonite, andbeidellite (formula 2); a trioctahedral type such as hectorite (formula3, preferably formula 8), saponite (formula 4, preferably formula 9),and nontronite; and the like.

[0177] The hydrotalcite is a layered mineral represented by the aboveformula 7, specifically Mg₆Al₂(OH)₁₆CO₃.4H₂O in which part of Mg²⁺ ofMg(OH)₂ (brucite having a laminate structure consisting of oxygenoctahedral layers having Mg²⁺ at the center) is substituted by Al³⁺isomorphously. The hydrotalcite has a positive charge, maintainselectrical neutrality by CO₃ ²⁻ between layers and has anionexchangeability. It is not a silicate mineral but is often treated as aclay mineral.

[0178] The compositions of some of the above-described layered inorganiccompounds used in the present invention are shown in Table 1 below.TABLE 1 Name of mineral Composition* Kaolinite Si₂Al₂O₅(OH)₄ HalloysiteSi₂Al₂O₅(OH)₄.2H₂O Serpentine Si₂(Mg²⁺,Fe²⁺)₃O₅(OH)₄ Talc Si₄Mg₃(OH)₂O₁₀Pyrophyllite Si₄Al₂(OH)₂O₁₀ MontmorilloniteMI_(x)Si₄(Al_(2−x)Mg_(x))O₁₀(OH)₂.nH₂0 BeidelliteMI_(x)(Si_(4−x)Al_(x))Al₂O₁₀(OH)₂.nH₂0 Hectorite MI_(x)Si4(Mg_(3−x)Li_(x))O₁₀(OH,F)₂.nH₂0 SaponiteMI_(x)(Si_(4−x)Al_(x))Mg₃O₁₀(OH)₂.nH₂0 NontroniteMI_(x)(Si_(4−x)Al_(x))Fe₂O₁₀(OH)₂.nH₂0 VermiculiteMI_(x)(Si_(4−x)Al_(x))Al₂O₁₀(OH)₂.nH₂0 Hydrotalcite Mg₆Al₂(OH)₁₆CO₃.H₂O

[0179] The average particle diameter of the layered inorganic compoundused in the present invention is not particularly limited if it is smallenough to enable the uniform dispersion of the compound. Since thelayered inorganic compound is generally a planer particle and hasdynamic equilibrium that the aggregation and cleavage of severalparticles are repeated, it is difficult to define the average particlediameter thereof. It is not easy to specify a preferable averageparticle diameter range. Daring to say, the value measured by means suchas light scattering method or observation through an electron microscopeis preferably 1 nm or more and 20 μm or less, more preferably 10 nm ormore and 2 μm or less when the layered inorganic compound is dispersedin water. Since the layered inorganic compound has ion exchangeability,it is considered that the layered inorganic compound adsorbs accordingto the charge and polarity of a dyestuff or the like. The ionexchangeability of the layered inorganic compound is derived from layercharge produced by the substitution of metal ions constituting a layer.Then, the absolute value of layer charge is preferably about 0.2 to 1for groups of atoms of the compositions of the formulas shown in Table1.

[0180] A layered inorganic compound containing transition metal ionssuch as iron ions as substituent ions in the structure or impurities iscolored by the metal ions and further shows oxidizing/reducingproperties and produces a side reaction with the result that it has poortransparency. Therefore, the layered inorganic compound is preferablynot substituted by transition metal ions, but the present invention isnot limited by this.

[0181] In the above layered inorganic compounds such as clay minerals,the distance, charge and polarity between layers can be adjusted inadvance by establishing a pillar such as a quaternary ammonium salt.

[0182] Out of the above layered inorganic compounds used in the presentinvention, more preferred are 2:1 type clay minerals and particularlypreferred are swelling clay minerals having ion exchangeability.

[0183] Out of the swelling clay minerals, more preferred are bentonite,smectite, vermiculite and synthetic fluorine mica, and particularlypreferred are synthetic smectite such as synthetic hectorite andsynthetic saponite, and synthetic mica (natural mica is generally anon-swelling clay mineral) such as swelling synthetic mica (Na typemica) typified by synthetic fluoro-mica. The swelling function of theswelling clay minerals is derived from an exchangeable cation or anion.A swelling layered inorganic compound is preferably used because thedetectable substance is quickly adsorbed by the surface of theinterlayers and the surface of a clay association called card house.Clay minerals may adsorb anionic substances, catibnic substances andnonionic polar organic compounds, and hydrotalcite may adsorb anioniccompounds. Compounds which can be adsorbed by the layered inorganiccompounds are detailed in Chapter 11 “Interaction of Clays and OrganicCompounds” of “An Introduction to Clay Colloid Chemistry, SecondEdition” written by H. Van Olphen (Krieger Publishment, Malabar). Theymay be used alone or in combination of two or more in the presentinvention.

[0184] The above layered inorganic compound used in the presentinvention may be either synthetic or natural but is preferablysynthetic. Unlike natural layered inorganic compounds, synthetic layeredinorganic compounds is chemically uniform and make it possible toquantitatively handle the detectable substance which has been adsorbedand quantitatively and optically handle the detectable substance becausethey do not contain colored metals such as iron between layers andaccordingly, have high transparency. “Synthetic” as used herein meansthat layered inorganic compounds are produced mainly by a hydrothermalsynthetic method or melting method at least in the case of smectite.Swelling clay minerals obtained by purifying natural products are alsopreferably used.

[0185] These layered inorganic compounds are commercially available.Examples thereof include Lucentite SWN and Lucentite SWF (synthetichectotite) and ME (fluoro-mica) of Co-op Chemical Co., Smecton SA(synthetic saponite) of Kunimine Kogyo Co., Thixopy W (synthetichectorite) and Kyoword 500 (synthetic hydrotalclte) of Kyowa ChemicalIndustry Co., Raponite (synthetic hectorite) of Laporte Co., naturalbentonite marketed by Nacalai Tesque Co., Multigel (bentonite) ofToyojun Kogyo Co., and the like (Names with captial initials are tradenames.).

[0186] It is known that the above layered inorganic compounds adsorborganic compounds such as amines, polyenes and various dyestuffs. Theyhave been used as a water treatment agent which adsorbs oil, dyestuffsand the like, a protein removing agent used in the production of wine,sweet sake and the like, as a decoloring and purifying agent whichadsorbs and removes impurities, and the like. These layered inorganiccompounds are known as a substance providing a specific reaction site bycausing a phenomenon called “metachromasy” and further known as asubstance improving the optical stability of a natural dyestuff inrecent years.

[0187] However, in the first method of the present invention, it hasbeen found that measurement sensitivity can be increased by allowing thedetectable substance to be adsorbed by the layered inorganic compound.Therefore, by the addition of the layered inorganic compound, themeasurement of hydrogen peroxide in the above reaction system using 4-AAand a hydrogen donor, for example, can be carried out morequantitatively. A case in which the sensitivity of measurement isincreased by using a layered inorganic compound such as a clay mineralin the measurement of a substance has not been reported yet.

[0188] In the second method of the present invention, an attempt hasbeen made to add these layered inorganic compounds to a reaction systembased on the assumption that there are such effects that a complex isformed by the adsorption of the detectable substance to the layeredinorganic compound, the detectable substance is protected from thereaction system, and the electron level which involves in adecomposition reaction is changed by adsorption. As a result, it hasbeen found that the detectable substance is adsorbed by the layeredinorganic compounds and the adsorbed detectable substance can existfully stably in the presence of excessive hydrogen peroxide or ascorbicacid. By the addition of the layered inorganic compound, the measurementof hydrogen peroxide in the above reaction system using 4-AA and ahydrogen donor, for example, can be carried out with higher accuracy. Acase in which a layered inorganic compound such as a clay mineral isadded to a reaction system for measuring an analyte, to stabilize theformed detectable substance and improve the sensitivity and accuracy ofthe measurement of the analyte has never been reported yet.

[0189] In the third method of the present invention, it has been foundthat the formation reaction of the detectable substance is allowed toproceed in the presence of the layered inorganic compound for thepurpose of analysis, and though its mechanism is not fully known, thereaction precursor of the detectable substance is adsorbed by thelayered inorganic compound and concentrated on the surface of thelayered inorganic compound to improve the rate of the formation reactionof the detectable substance, thereby making it possible to quickenmeasurement.

[0190] In the fourth method of the present invention, it has been foundthat even if an insoluble substance is formed, high-sensitivity quickdetection can be made by dispersing the layered inorganic compound inthe solvent of the reaction system including the formation reaction ofthe detectable substance. A case in which the layered inorganic compoundsuch as a clay mineral is dispersed in a solvent to measure a substancehas never been reported yet.

[0191] More surprisingly, measurement accuracy is not impaired by theaddition of a layered inorganic compound as a detection reaction is notinterrupted even when the layered inorganic compound is caused to existin a reaction system.

[0192] A method for causing the layered inorganic compound to exist inthe reaction system in the method of the present invention, depends onthe reaction system used, and preferably, the layered inorganic compoundis dispersed in the reaction medium of the reaction system in the formof any one selected from dispersion, sol, gel, slurry, agglomerate,aggregate and sintered porous body. The reaction medium of the reactionsystem may be the reaction solvent of the formation reaction of thedetectable substance. In the fourth method of the present invention, itis the reaction solvent of the formation reaction of a substanceinsoluble in the reaction solvent.

[0193] To cause the layered inorganic compound to exist in the reactionsystem, more preferably, the layered inorganic compound is dispersed ina solvent and the resulting dispersion is added to the reaction system.The solvent is not particularly limited and any conventionally knownsolvents may be used. Examples of the solvent include water such asdistilled water, alcohols such as ethanol, ketones such as acetone,ethers such as diethyl ether, esters such as ethyl acetate, halogenatedhydrocarbons such as chloroform, aromatic hydrocarbons such as benzeneand toluene, and the like, from which one suitable for an analyte and adetection reaction system thereof can be selected. Preferably, thelayered inorganic compound is dispersed in a buffer which will bedescribed hereinafter and the resulting buffer dispersion is added tothe reaction system. It is known in dry chemistry that a sample solutionsuch as blood, saliva and urine containing an analyte can be used as areaction solvent.

[0194] The amount of the layered inorganic compound added is determinedaccording to a reaction system used. Depending on the layered inorganiccompound used, it is preferably such as to eliminate a case where thenumber of adsorption sites is too small for a detectable substance andthe detectable substance cannot be adsorbed completely and remains inthe solution, or a case where the number of adsorption sites is toolarge and there are differences in the concentration of the detectablesubstance adsorbed at these sites.

[0195] The preferable amount of the layered inorganic compound added tothe reaction system is determined as follows. That is, since the layeredinorganic compound adsorbs a dyestuff or the like in an amountcorresponding mainly to the degree of the above layer charge, the totalnumber of adsorption sites of the dyestuff or the like can be obtainedfor each type of layered inorganic compound. When the concentration of areagent in a detection reaction system is determined, the approximatemaximum amount of a formed dyestuff or the like can be calculated, andeach type of layered inorganic compound can be added so that the maximumamount of the dyestuff or the like which can be formed does not exceedthe whole adsorption sites of the layered inorganic compound.

[0196] The time for adding the layered inorganic compound is notparticularly limited and may be before or after the formation reactionof the detectable substance. Preferably, the layered inorganic compoundis added to a reaction system before the formation react-ion of thedetectable substance and dispersed in the reaction system.

[0197] The reason why the layered inorganic compound is dispersed in thereaction system is that interaction such as adsorption between thereaction starting substance, the reaction intermediate or the reactionproduct involved in the formation reaction of the detectable substanceand the layered inorganic compound can readily occur. Another reason isthat a state where the layered inorganic compound is uniformly dispersedwithout differences in concentration is suitable for detection.Therefore, the term “dispersion” as used herein means a state where thelayered inorganic compound is dispersed in a solution or a state of asol or gel, or a state suitable for detection where the aboveinteraction readily occurs.

[0198] In the fourth method of the present invention, an insolublesubstance can be adsorbed by the layered inorganic compound by uniformlydispersing the layered inorganic compound in the reaction solvent tomake it existent in the reaction system.

[0199] The dispersion medium in which the layered inorganic compound isdispersed is not always the same as a reaction solvent in which areaction takes place. The layered inorganic compound may be dispersed ina dispersion comprising a reaction solvent as a dispersion medium, or asol, a gel, an agglomerate, an aggregate or a sintered porous body whichthe reaction solvent can permeate. The dispersion medium is notparticularly limited if the layered inorganic compound can be uniformlydispersed therein.

[0200] The layered inorganic compound in the form of sol, gel,agglomerate, aggregate or sintered porous body which the reactionsolvent can permeate can be used as a detection portion which alsoserves as a reaction portion in a testing piece in dry chemistry or thelike. If the layered inorganic compound can be uniformly dispersed, themeasuring method of the present invention can be applied to the testingpiece.

[0201] When the layered inorganic compound having an exchangeable cationor exchangeable anion is dispersed in water by agitation orultrasonication, an uniform dispersion can be obtained if it has anappropriate concentration. However, by the addition of an electrolyte oran organic compound or by long-time standing or temperature variations,the particles of the layered inorganic compound may agglomerate,aggregate or form a gel or precipitate. The agglomeration is generallycaused by the gentle interaction of the particles and the particles canbe re-dispersed by agitation easily.

[0202] The dispersion, agglomeration and re-dispersion of the layeredinorganic compound are detailed, for example, in Chapter III “The Theoryof Stability of Hydrophobic Sols”, Chapter IV “Successes of the Theoryof Stability—Further Theories and Refinement”, Chapter VII “ElectricDouble-Layer Structure and Stability of Clay Suspensions” and ChapterVIII “Peptization of Clay Suspensions” of “An Introduction to ClayColloid Chemistry, Second Edition” written by H. Van Olphen (KriegerPublishment, Malabar).

[0203] The degree of adsorption is influenced by the composition of abuffer (pH, ionic strength, components forming a complex, and the like).For example, smectite dispersed in purified water hardly adsorbsbrilliant blue FCF whereas smectite dispersed in a bis-tris buffersolution having a pH of 6.5 [prepared frombis(2-hydroxyethyl)iminotris(hydroxymethyl)methane and hydrochloricacid] quickly adsorbs this dyestuff.

[0204] Examples of the buffer or buffer solution used in the method ofthe present invention include a phosphoric acid buffer solution, citricacid buffer solution, N-(2-acetoamide)imino diacetic acid buffersolution and the like, besides the above bis-tris buffer solution. Thepresent invention is not limited to these and a suitable one is suitablyselected according to the reaction system used. The pH, concentrationand the like of the buffer are preferably suitably selected according tothe reaction system used.

[0205] The time for adding the buffer is not particularly limited andmay be before or after the addition of the layered inorganic compound.It is preferably added as a buffer solution containing the layeredinorganic compound dispersed therein to a reaction system together withthe layered inorganic compound.

[0206] 4. Surfactant

[0207] In measurement with the method of the present invention, varioussurfactants can be added to the reaction system. Addition of asurfactant makes it possible to uniformly disperse a sample containing ahardly soluble substance and uniformly and quickly infiltrate the sampleinto the test portion of a testing piece by improving the wettability bythe sample. Since the surfactant has a function to disperse or dissolvea substance adsorbed to the interface, it conflicts with the adsorptionof the formed detectable substance to the layered inorganic compound ordissolve the formed detectable substance. Therefore, it may weaken theeffect of the present invention. As the surfactant used in combinationwith the layered inorganic compound in the present invention, asurfactant which does not interfere the adsorption of the formeddetectable substance to the layered inorganic compound is preferablyselected. The amount of the surfactant used is preferably small enoughto prevent the interference of the adsorption. To adjust the adsorptionstrength between the detectable substance such as a dyestuff and thelayered inorganic compound, a known surfactant suitable for the reactionsystem may be used and the amount of the surfactant added may becontrolled.

[0208] The type of surfactant which does not interfere the adsorptionmay be one of which molecular weight is not much larger than that of theformed dyestuff and the organicity and the inorganicity of which satisfythe following equation:

(inorganicity)=(2.37±0.23)×(organicity)−186.2±117.1

[0209] The above equation is obtained by studying the adsorptioninterference effect and the relationship between the inorganicity andthe organicity of each of various surfactants having known structures.That is, the number of points is allotted to each functional group oratom, for example, 20 is allotted to a single carbon atom as anorganicity, 100 to a hydroxyl group as an inorganicity, 30 topolyethylene oxide as an organicity and 60 to the polyethylene oxide asan inorganicity, 70 to a nitro group as an organicity and 70 to thenitro group as an inorganicity. Then, the total of inorganicity valuesand the total of organicity values are obtained by summing the numbersof points for functional groups and atoms constituting a compound. Thereis known an organic conceptional diagram in which a total inorganicityand a total organicity are plotted on rectangular coordinates, compoundshaving similar properties are located in the same area of therectangular coordinates, and common properties appears without dependingon the structure of a compound (Yoshio Kohda, “Organic ConceptionalDiagram—Basis and Application-”, p. 11, Sankyo Shuppan (1984)). Theinventors of the present invention have studied the adsorptioninterference effects and the relationship between the inorganic valuesand organic values of many surfactants having known structures and havefound that surfactants which do not interfere the adsorption satisfy theabove equation in the organic conceptional diagram. Although calculationdata on the above organic conceptional diagram in the above book can beused for the calculation of the inorganicity and the organicity, theabove equation was obtained from calculation data provided in “PersonalComputer Organic Conceptional Diagram” program manufactured by Dr.Yoshio Honma (The Chemical Software Society of Japan and the like).

[0210] The type and amount of a surfactant which does not interfereadsorption can be selected as follows.

[0211] (1) Hydrogen peroxide is added to a reaction solution containinga predetermined amount of smectite, 4-AA andN-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline to developcolor.

[0212] (2) A surfactant is added to a reaction solution having he samecomposition as in (1) above to the same concentration and hydrogenperoxide is added to develop color.

[0213] (3) Smectite is separated by appropriate means such as settling,centrifugal sedimentation or filtration, the color tone of thesupernatant or filtrate is measured by a spectrophotometer, and theamounts of the formed dyestuff absorbed by the smectite are comparedbetween (1) and (2). Alternatively, when flocculation caused byadsorption is observed, the amount of adsorption is evaluated with thedegree of flocculation or settling.

[0214] (4) The type and amount of a surfactant which does not produceany difference between when it is added and when it is not added areselected.

[0215] Preferred examples of the surfactant which is selected by theabove methods include sugar-alkyl ethers such asn-octyl-β-D-glucopyranoside; sugar-alkyl thioethers such asn-octyl-β-D-thioglucopyranoside and n-heptyl-β-D-thioglucopyranoside;super amides such as n-octanoyl-N-methylglucamide andn-nonanoyl-N-methylglucamide, sugar esters such asβ-D-fructopyranosyl-α-D-glucopyranoside monodecanoate;N,N-bis(3-D-gluconamidepropyl)deoxycolamide and the like.

[0216] The amount of the surfactant added is not particularly limitedand the proportion of the surfactant to the total amount of the layeredinorganic compounds is not particularly limited as well. An amountsuitable for the types of the surfactant and the layered inorganiccompound and the reaction system may be selected. The surfactant is usedin an amount that its effect can be exhibited sufficiently, for example,an amount which does not greatly exceed the critical micellconcentration of the surfactant used in an aqueous solution. Forexample, a 0.3% aqueous solution of n-octyl-β-D-thioglucopyranoside, a0.3% aqueous solution of β-D-fructopyranosyl-α-D-glucopyranosidemonodecanoate and a 0.3% aqueous solution ofN,N-bis(3-D-gluconamidepropyl)deoxycolamide are preferably used.

[0217] The surfactant is preferably used particularly when measurementis carried out by the first to third methods of the present invention.

[0218] 5. Embodiments of Measuring Method

[0219] In a preferred embodiment of the measuring method of the presentinvention, the layered inorganic compound is added to the reactionsystem and dispersed in the reaction system in advance. In thedispersion, a translucent colloidal agglomerate may be produced but theagglomerate does not always need to be produced in the presentinvention. This agglomerate can be considered as a complex comprisingthe layered inorganic compound and the detectable substance adsorbedthereby. This agglomerate may be uniformly re-dispersed by stirring.When this agglomerate is inconvenient, the dispersibility of the layeredinorganic compound is improved by using a phosphate buffer solution,thereby making it possible to suppress the production of an agglomerate.

[0220] It is also possible to further improve measurement sensitivity byallowing the detectable substance used finally for detection to beadsorbed by the layered inorganic compound, thereby settling it, andseparating the detectable substance from the reaction system andconcentrating it. The method for separating the layered inorganiccompound by which the detectable substance has been adsorbed is notparticularly limited and selected from, for example, settling,centrifugation, filtration, chromatography, electrophoresis, solventevaporation and the like. Specifically, the filtration of a dispersionof the layered inorganic compound exemplified in the present inventioncan be carried out, for example, by using a polysulfone ultrafiltrationmembrane having an exclusion limit of about 10,000 or a pore size ofabout 5 nm.

[0221] In the present invention, the detectable substance adsorbed bythe layered inorganic compound is measured. The measuring method may beselected from an absorption measuring method, fluorescence measuringmethod, luminescence measuring method, electrochemical measuring method,light scattering measuring method, reflectance measuring method or thelike. Preferably, it is an optical measuring method such as colorimetrytypified by absorptiometry using an absorptiometer or the like. Sincethe layered inorganic compound used in the present invention rarelyabsorbs light of a visible to near infrared range, even in the form ofcolloidal dispersion or gel, optical measurement can be carried out.When the dispersion is measured directly, such means as an opal glassmethod can be selected as the system. Since a porous structure which areaction solvent can permeate can be fabricated using the layeredinorganic compound as described later, a testing piece having thisportion as a detection portion which also serves as a reaction portionis used to carry out reflectance measurement, absorption measurement,fluorescence measurement and the like. An electrochemical measuringmethod for measuring an oxidation/reduction current or a membranepotential with an electrode can also be used. The electrode is contactedto the layered inorganic compound by which the detectable substance hasbeen adsorbed to measure electrochemical response with high sensitivity.

[0222] II. Testing Piece of the Invention

[0223] The testing piece of the present invention is an analyticaltesting piece for measuring an analyte by measuring a detectablesubstance which is formed when the analyte contained in a sample reactswith a reagent. The testing piece comprises at least one test portionhaving a detection portion for detecting the detectable substance.

[0224] The test portion is a functional portion responsible for a seriesof analytical processes of the absorption, diffusion, reaction,detection and the like of the sample in the testing piece. The testportion, whose structure is not particularly limited, generallycomprises the detection potion for detecting the detectable substancesuch as a dyestuff with reflectance, transmission/absorption,fluorescence or the like; a sample suction portion, provided at the endor near the end of the test portion, for sucking and introducing thesample into the test portion; a diffusion/infiltration portion foruniformly infiltrating and diffusing the sample into the test portion; areagent portion containing a reagent which reacts with the analytecontained in the sample; a reaction portion where a reaction such as adetection reaction takes place; a developing portion for separating acomponent contained in the sample or a dyestuff formed by the detectionreaction by a function similar to chromatography; a time control portionfor controlling the proceeding of a reaction by utilizing the timeduring which the sample moves; a holding portion for trapping orremoving a component contained in the sample or the formed dyestuff byan adsorption function; an absorption portion, provided at the end ornear the end of the test portion on a side opposite to the samplesuction portion with respect to the detection portion, for absorbingexcess of a sample solution, a washing solution and a developingsolution to prevent a back flow; and the like.

[0225] These portions bearing the functions of the test portion mayoverlap with one another in function. A single portion may have aplurality of functions, for example, the detection portion may alsoserve as the reagent portion and the reaction portion, and the detectionportion may also serve as the holding portion.

[0226] A preferred embodiment of the testing piece of the presentinvention is a testing piece comprising at least one multi-layered testportion composed of two or more layers including a detection layer fordetecting the detectable substance as the detection portion. Layersother than the detection layer include a sample suction layer forsucking a sample and introducing it into the test portion; a diffusionlayer for uniformly infiltrating and diffusing the sample into the testportion; a reagent layer containing a reagent which reacts with theanalyte contained in the sample; a reaction layer where a reaction suchas a detection reaction takes place; a developing layer or holdinglayer, formed between the reaction layer and the detection layer, havinga function to remove an interfering component; an absorption layer forabsorbing excess of the sample, a washing solution or a developingsolution added to prevent a back flow; an adhesive layer for fixing thetest portion on the support; and the like. Particularly preferably, itis a testing piece comprising a diffusion layer for diffusing the samplein addition to the detection layer so that the sample passes through thediffusion layer to be diffused and reaches the detection layer. Thetesting piece of the present invention may be a testing piece having asingle test portion or a multi-item testing piece having two or moretest portions. In the case of the multi-item testing piece, a pluralityof samples can be analyzed at the same time, and two or more analytescontained in the sample can be analyzed at the same time by usingdifferent reagents for different items.

[0227] Another preferred embodiment of the testing piece of the presentinvention is a testing piece comprising at least one test portion havinga detection area for detecting the detectable substance as the detectionportion. On the testing piece can be formed areas other than thedetection area, such as a sample suction area for sucking andintroducing a sample into the test portion; a diffusion area foruniformly infiltrating and diffusing the sample into the test portion; areagent area containing a reagent which reacts with an analyte containedin the sample; a reaction area where a reaction such as a detectionreaction takes place; a developing area for separating a componentcontained in the sample or a dyestuff formed by a detection reaction bya function similar to chromatography such as adsorption or distribution;a time control area for controlling the proceeding of a reaction usingthe time during which the sample moves; a holding area for trapping orremoving a component contained in the sample or the formed dyestuff byan adsorption function; an absorption area for absorbing excess of asample solution, a washing solution or a developing solution to preventa back flow; and the like. Particularly preferably, it is a testingpiece which comprises a diffusion area for diffusing the sample inaddition to the detection area so that the sample dropped onto the endor the like of the testing piece can pass through the diffusion area,move over the plane of the testing piece mainly by a capillaryinfiltration action and reach the detection area. In this case, thedetection area may have the above multi-layer structure composed of twoor more layers including the detection layer for detecting thedetectable substance. The testing piece of the present invention may bea testing piece comprising only one test portion composed of one set ofthe detection area and the reagent area, or a multi-item testing piececomprising two or more test portions thereon. In the case of themulti-item testing piece, a plurality of samples can be analyzed at thesame time, and two or more analytes contained in the sample can beanalyzed at the same time by using different reagents for differentitems.

[0228] In the present invention, the reaction portion where the analytecontained in the sample reacts with the reagent may be providedseparately from the detection portion so that the detectable substanceis formed in the reaction portion and then introduced and detected inthe detection portion. In this case, the detection portion is preferablyprovided at a position which the sample reaches after it is diffused andpasses through the reaction portion. Specifically, the detection layeris preferably formed at a position which the sample reaches after itpermeates from the surface of the multi-layered test portion, passesthrough the diffusion layer to be diffused, moves to the reaction layeras an intermediate layer and passes through the reaction layer. Thedetection area, the reaction area and the diffusion area are formed onthe testing piece, and the detection area is preferably formed in anarea which the sample reaches after it moves over the plane, permeatesthrough the diffusion area, moves to the reaction area and passesthrough the reactiop area.

[0229] In the present invention, the detection portion may also serve asthe reaction portion where the analyte contained in the sample reactswith the reagent so that the detectable substance can be formed by areaction between the analyte contained in the sample and the reagent inthe detection portion.

[0230] The detection portion in the present invention is a portion wherethe detectable substance such as a dyestuff formed by a reactionbetween.the analyte contained in the sample and the reagent is actuallydetected. However, the detection portion may also serve as the reactionportion where the above reaction takes place or the reagent portioncontaining the reagent as described above. In this case, the reagent isgenerally contained in the detection portion in advance. Meanwhile, inthe present invention, the testing piece may have a detection portionindependent from the reaction portion and the reagent portion. In thiscase, the reagent may not be contained in the test portion and may beadded before and/or after the addition of the sample. Or, a solution ofthe detectable substance such as a dyestuff formed by a reaction betweenthe analyte and the reagent may be added.

[0231] The testing piece of the present invention generally comprisesthe test portion and a support portion shaped like a sheet, a tube or arod for supporting the test portion, and optinally a.sensor such as anelectrode, a sample solution suction device and the like.

[0232] The present invention is preferably applied to a testing piecewhich uses a reagent which can form the detectable substance such as adyestuff and the reaction system as described hereinafter.

[0233] The reagent is not particularly limited if it can form a complexby interaction such as adsorption between the detectable substance suchas a dyestuff formed by a reaction and the layered inorganic compoundused in the present invention as described in the section of themeasuring method of the present invention.

[0234] The reagent for forming the detectable substance to be adsorbedby the layered inorganic compound or the like can be widely found incompounds such as a dyestuff precursor for forming an opticallydetectable substance such as a dyestuff or a fluorescent dyestuffthrough an oxidation-reduction reaction, an acid-base reaction, acondensation reaction, a complex formation reaction and the like,compounds for forming the electrochemically detectable oxidized/reducedform of an electron carrier or a complex compound, and the like.

[0235] Since the sample, the reagent or the reaction product is often inthe form of a solution comprising water as a solvent, if the detectablesubstance is water-soluble, it is readily diffused and eluted.Therefore, when the detectable substance is water-soluble, the effect ofthe present invention is particularly marked. Therefore, the usedreagent is preferably a reagent for forming a water-soluble detectablecompound. In fact, a large number of such reagents are used. However,the reagent is not limited to these. The sample, the reagent or thereaction product may be dissolved in a solvent other than water. In thiscase, the used reagent may be a reagent for forming a detectablesubstance which is diffused and eluted by that solvent. A reagent forforming a detectable substance insoluble in the solvent of the sample,the reagent or the reaction product may be used.

[0236] Any reagents are acceptable if they form detectable substances aslisted in the description of the measuring method of the presentinvention. Preferred examples of the reagent include compounds having aconjugate system such as an aromatic ring, specifically reagents (whichform quinone dyestuffs when oxidation-condensed) for a coupler typifiedby 4-amino-1,2-dihydro-1,5-dimethyl-2-phenyl-3H-pyrazol-3-one and ahydrogen donor (N-ethyl-N-(3-sulfopropyl)-3,5-dimethylaniline and thelike) as dyestuff precursors; dyestuff precursors for formingcolor-producing dyestuffs by oxidation such as o-tolidine and benzidines(3,3′,5,5′-tetramethylbenzidine and the like); leuco substances(developing color when oxidized) of dyestuffs such as2,6-dichloro-4-[(4-hydroxyphenyl)imino]-2,5-cyclohexadien-1-one;compounds for forming fluorescent substances when oxidized, such as4-hydroxyphenylacetic acid; luminescent substances such aschemiluminescent substances; reagents for forming dyestuffs when reducedsuch as tetrazolium salt (forming formazan when reduced) and1,1°-dimethyl-4,4′-bipyridinium salt; compounds which develop color orchange their colors by pH variations, such as Bromocresol Green; knownreagents for coloration reactions such as diazonium salts (forming azodyestuffs by coupling) including 2-methoxy-4-morpholinobenzenediazoniumsalt and 2,3-dimethyl-2,3-bis(hydroxyamino)butane (developing color whenreacting with aldehyde); known reaction reagents such aso-phthalaldehyde (forming a fluorescent substance when reacting withhistamine); enzyme substrates such as 4-methylumbelliferyl phosphate;compounds which develop color or change their colors by forming acomplex such as2-(5-bromo-2-pyridylazo)-5-[N-propyl-N-(3-sulfopropyl)amino]anilinesalt; and compounds capable of forming the above detectable substances.

[0237] Reaction systems for forming the above detectable substances arethose used in the measuring method as described in the section of themeasuring method of the present invention. The reaction systems includethe following.

[0238] (a) A reaction system including a reaction for forming hydrogenperoxide or an oxidation reaction using hydrogen peroxide as anoxidizing agent.

[0239] (b) A reaction system including a reaction for formingnicotinamide adenine dinucleotide (NADH) or nicotinamide adeninedinucleotide phosphate (NADPH) or a reaction in which NADH or NADPHfunctions as a reducing agent.

[0240] (c) A reaction system using a reaction for forming a diazoniumsalt by allowing nitrous acid to react with an aromatic primary amine inthe presence of an acid.

[0241] (d) A reaction system including a reaction in which a fluorescentenzyme substrate such as 4-methyl-umbelliferone having a phosphoric acidester forms a fluorescent substance by separating a phosphate by thefunction of alkali phosphatase.

[0242] (e) A reaction system including a reaction for forming anoxidized/reduced form of an electron carrier by oxidizing/reducing theelectron carrier such as 1,4-diaminobenzene with an oxidizing/reducingenzyme.

[0243] Analytical methods using the above reaction systems includeimmunoassay such as ELISA, immunochromatography, urine examination,biochemical blood examination, colorimetry and the like. Testing piecesused in these analytical methods are detailed in such documents as H. G.Curme, et al., Clinical Chemistry, 24 (8), 1335-1342 (1978); B. Walter,Analytical Chemistry, 55 (4), 498A (1983); Asaji Kondo, Bunseki, 1984(7), 534; Asaji Kondo, Bunseki, 1986 (6), 387; Bunseki Kagaku Binran, p.8 (edited by the Japan Society for Analytical Chemistry: fourth revisededition, Maruzen (1991)); Japanese Patent Application Laid-open No.6-213886(1994) (Masao Kitajima, et al.); M. P. Allen, et al., ClinicalChemistry, 36 (9), 1591-1597 (1990); D. Noble, Analytical Chemistry, 65(23), 1037A (1993); R. F. Zuk, et al., Clinical Chemistry, 31 (7),1144-1150 (1985) and the like. Analytes which can be analyzed by thesemethods include biological components contained in the body fluid suchas urine and blood, trace amounts of substances existent in foods,medicines or natural environment, industrial chemical substances, traceamounts of substances contained in waste, and the like. The testingpiece of the present invention can be used in the analysis of thesesubstances.

[0244] A sample to which the testing piece of the present invention canbe applied may contain only one analyte or two or more analytes.

[0245] A known compound which is generally used in an analytical testingpiece, such as a hydrophilic polymer, can be contained in the testportion of the testing piece of the present invention as required.

[0246] In the present invention, a layered inorganic compound must becontained in the test portion of the testing piece, preferably thedetection portion which is a portion where a formed dyestuff exists ofthe test portion.

[0247] That is, the layered inorganic compound must be contained in atleast one detection layer or detection area constituting the testportion. Specifically, in a multi-layered test portion composed of twoor more layers including the detection layer, the layered inorganiccompound is contained at least in the detection layer. In this case, thelayered inorganic compound may be further contained in a layer otherthan the detection layer, for example, the sample suction layer, thediffusion layer, the reagent layer, the reaction layer, the adhesivelayer, the holding layer, the developing layer, the absorption layer orthe like.

[0248] When the test portion has the detection area, the layeredinorganic compound is contained at least in the detection area. Further,the layered inorganic compound may be further contained in an area otherthan the above detection area, for example, the sample suction area, thediffusion area, the reagent area, the reaction area, the developingarea, the time control area, the holding area, the absorption area orthe like. In this case, the detection area may have a multi-layerstructure. In this case, the layered inorganic compound is contained atleast in the detection layer out of layers constituting the detectionarea. The layered inorganic compound may be further contained in otherlayer.

[0249] When the test portion has a reaction portion where an analytecontained in a sample reacts with a reagent besides the detectionportion, the detection portion is preferably provided at a positionwhich the sample reaches after it is diffused and passes through thereaction portion so that the detectable substance formed in the reactionportion moves to the detection portion containing the layered inorganiccompound to be detected.

[0250] Examples of the layered inorganic compound used in the testingpiece of the present invention include those listed in the abovedescription of the measuring method of the present invention. Like themeasuring method of the present invention, out of these layeredinorganic compounds, preferred are 0.2:1 type clay minerals andparticularly preferred are swelling clay minerals having ionexchangeability. Out of the swelling clay minerals, more preferred arebentonite, smectite, vermicullite, and synthetic mica (natural mica isgenerally a non-swelling clay mineral) such as swelling synthetic mica(or Na type mica) typified by synthetic fluoro-mica, and particularlypreferred are synthetic smectite such as synthetic hectorite andsynthetic saponite, and synthetic fluoro-mica. They may be used alone orin combination of two or more. No attempt has been made so far tocontain a layered inorganic compound in a testing piece utilizing itseffect of suppressing the diffusion or elution of a dyestuff or thelike.

[0251] Surprisingly, when the layered inorganic compound is added to thetest portion such as the detection portion, a detection reaction is notinterfered. Thereby, addition of this layered inorganic compound makesit possible to carry out examination making use of, for example, theabove reaction system using 4-AA and a hydrogen donor more accuratelyand easily without being worried by elution.

[0252] A portion containing the layered inorganic compound of the testportion is preferably a porous structure, though its substance is notparticularly limited, but it is preferably formed mainly by the layeredinorganic compound, or the layered inorganic compound and at least oneporous substance selected from the group consisting of hydrophilicpolymers, membrane filters, fiber assemly such as filter paper, cloth,and glass filters, and organic and inorganic compound fine powders suchas cellulose and diatomaceous earth.

[0253] The porous structure formed by the layered inorganic compound isa sol, a gel, an agglomerate or aggregate of the layered inorganiccompound, or a porous body obtained by drying or sintering these. Abuffer to be described later may be added to the porous structure. Forexample, a drop of a 1% dispersion of the layered inorganic compound islet fall upon a support, cast and lyophilized to obtain an absorptiveporous layer.

[0254] The support may be shaped like a sheet, a tube or a rod. Thematerial of the support is not particularly limited and may be selectedfrom fiber assembly such as filer paper, nonwoven cloths, cloths andglass filters; granular substances such as glass beads, polymer beadsand titanium dioxide; granular substances and fine powders such ascellulose, diatomaceous earth, soluble salt and hydrophobicpolysaccharide powders; membrane filters; organic polymers such asplates of plastic including polyethylene terephthalate (PET) andpolystyrene; and the like. More preferably, the substance is a gel of ahydrophilic polymer, or a membrane filter or a plastic plate whosesurface is made hydrophilic.

[0255] The hydrophilic polymer may be a polymer, a copolymer, anassociated substance or the like containing a chemical structure such asan polyalkylene oxide exemplified by polethylene oxide and polypropyleneoxide; cellulose derivative exemplified by carboxymethyl cellulose andhydroxyethyl cellulose; gelatin or derivative thereof (such asphthalated gelatin); other polysaccharide or derivative thereof(agarose, carrageenan, chitin, chitosan or the like); polyvinyl alcohol;polyvinyl pyrrolidone; polyacrylate (sodium polyacrylate, copolymerthereof with maleic acid or the like); polyacrylamide; polymethacrylicacid (polyhydroxyethyl methacrylate or the like); methacrylamide;polysulfone; polyimide; polystyrene; polycarbonate; polyether etherketone; polyoxymethylene; sodium alginate; or polyolefin resinexemplified by polyethylene, polypropylene or polyfluoroethylene whichis made hydrophilic (by exposure to ultraviolet light, silanol treatmentor the like).

[0256] Preferably, the above hydrophilic polymer has a network structureproduced by graft polymerization using a crosslinking agent orassociation due to hydrophobic affinity and is insoluble in water.

[0257] Examples of the hydrophilic polymer include polylysinecrosslinked by glutaraldehyde, polyethylene oxide crosslinked product,polyacrylamide graft polymer, polyacrylate graft polymers,starch-acrylate graft polymers and the like.

[0258] At least one porous substance selected from the group consistingof hydrophilic polymers, membrane filters, fiber assembly and organicand inorganic compound fine powders and the layered inorganic compoundmay be both contained in the test portion to form the porous structure.To form this porous structure, a method in which a mixture solution of aporous structure forming substance and the layered inorganic compound isprepared and cast on or impregnated into the above-described support, amethod in which a porous structure forming substance is used to form aporous support such as a porous film and a dispersion or a mixturesolution of the layered inorganic compound is cast or impregnated intothe porous support, or the like may be used.

[0259] To mix the layered inorganic compound at the time of productionof the porous structure, for example, a method in which the layeredinorganic compound is kneaded with a hydrophilic polymer or fine powdersand formed into a film is used. Alternatively, a buffer solution havingthe layered inorganic compound dissolved or dispersed in a buffer to bedescribed later may be dried and the obtained dried product may be mixedwith a raw substance.

[0260] When the dispersion or the mixture solution of the layeredinorganic compound is impregnated into the porous support, the type ofsolvent used is not particularly limited and conventionally knownsolvents can be used. A solvent suitable for the detection reactionsystem used can be selected from water such as distilled water, alcoholssuch as ethanol, ketones such as acetone, ethers such as diethyl ether,esters such as ethyl acetate, halogenated hydrocarbons such aschloroform, and aromatic hydrocarbons such as benzene and toluene.Preferably, the porous support is impregnated with a buffer solutioncomprising the layered inorganic compound dissolved or dispersed in abuffer to be described later. The concentration of the solution ordispersion can be suitably selected according to a reaction system orthe like and is not particularly limited.

[0261] A method for forming a layer or an area containing the layeredinorganic compound will be described below.

[0262] To form the layer or the area containing the layered inorganiccompound, a porous structure obtained by drying or sintering a sol, agel, an agglomerate or an aggregate of the layered inorganic compoundcan be used. For example, a drop of a 1% dispersion of the layeredinorganic compound is cast on a plastic sheet, and lyophilized to obtaina porous layer having high absorptivity.

[0263] At least one porous structure forming substance selected from thegroup consisting of the above hydrophilic polymers, membrane filters,fiber assembly and organic and inorganic powders can be used in theformation.

[0264] The hydrophilic polymer is particularly preferably gelatin,polyacrylic acid or derivative thereof, polyacrylamide, polyvinylalcohol, polyvinylpyrrolidone, polyethylene glycol, polysaccharide orderivative thereof, polypeptide, or polyamine or derivative thereof. Thehydrophilic polymer maybe used as a gel or a dried product thereof. Thehydrophilic polymer may be a gel whose degree of crosslinking iscontrolled by the addition of a known crosslinking agent such asglutaraldehyde. These hydrophilic polymers may be used alone or incombination.

[0265] To obtain a porous structure comprising the above substance andthe layered inorganic compound, various methods already described in thesection of the method for containing the layered inorganic compound inthe test portion can be used. For example, a 1% dispersion of thelayered inorganic compound dispersed in a buffer solution is impregnatedinto filter paper and dried with hot air to obtain a porous area. Asmall piece of the filter paper is affixed to a plastic sheet to obtaina porous layer.

[0266] The following procedure, for example, can be employed. The sameamount of an aqueous solution of a polyacrylamide having a predeterminedconcentration is mixed with a 3% dispersion of the layered inorganiccompound in a polyacrylamide/layered inorganic compound weight ratio of1:1 to 4:1 and stirred well for several hours. pH of the resultingmixture solution is adjusted to about 5 to 9 by adding a diluted aqueoussolution of sodium carbonate or acetic acid if necessary. If necessary,the mixture solution is made alkaline, N,N-methylenebisacrylamide isadded to a concentration of 2%, and the mixture solution is exposed toelectron beams to cause a crosslinking reaction. The thus obtainedmixture solution is coated on a plastic plate and dried to obtain aporous film.

[0267] The thus formed porous structure containing the layered inorganiccompound is excellent particularly in absorptivity and preferably usedas the test portion of a testing piece. As a matter of course, theformation example of the test portion is not limited to this. Forexample, the formation of the test portions of various known testingpieces can be applied. These testing pieces are described in suchdocuments as H. G. Curme, et al., Clinical Chemistry, 24 (8), 1335-1342(1978); B. Walker, Analytical Chemistry, 55 (4), 498A (1983); AsajiKondo “Bunseki” 1984 (7), 534; R. F. Zuk, et al., Clinical Chemistry, 31(7), 1144-1150 (1985); Asaji Kondo, Bunseki, 1986 (6), 387; JapanesePatent Application Laid-open No. 2-6541(1990) (K. Hildenbrand); M. P.Allen, et al., Clinical Chemistry, 36 (9), 1591-1597 (1990); JapanesePatent Application Laid-open No. 3-163361(1991) (E. J. Kiser, et al.);Bunseki Kagaku Binran, p. 8 (edited by the Japan Society for AnalyticalChemistry: Fourth Revised Edition, Maruzen (1991)); D. Noble, AnalyticalChemistry, 65 (23), 1037A (1993); Japanese Patent Application Laid-openNo. 5-157745(1993) (Hidehiko Manabe, et al.); Japanese PatentApplication Laid-open No. 6-213886(1994) (Masao Kitajima, et al.);Japanese Patent Application Laid-open No. 6-222061(1994) (H. Brandt, etal.); and the like.

[0268] The concentration of a dispersion of the layered inorganiccompound, the mixing ratio thereof to the hydrophilic polymer and a pHvalue to be controlled are suitably selected based on parameters such asthe type of the layered inorganic compound, the type of a dyestuff to beadsorbed, the type and amount of the hydrophilic polymer used, the typeand amount of the buffer and the viscosity of the mixture solution so asto obtain a porosity, a film thickness of the porous layer and amechanical strength of the test portion which are required.

[0269] By adding a reagent which reacts with an analyte contained in asample solution to form the detectable substance to the porous structurethus formed, the porous structure can be used as the test portion of thetesting piece.

[0270] The amount of the layered inorganic compound added is determinedaccording to a reaction system used. Depending on the layered inorganiccompound used, it is preferably such as to eliminate a case where thenumber of adsorption sites is too small for the formed substance and theformed substance cannot be adsorbed completely and remains in thesolution, or a case where the number of adsorption sites is too largeand there are differences in the concentration of the formed substanceadsorbed at these sites, as in the measuring method of the presentinvention. As for the preferred amount of the layered inorganic compoundto be added to the reaction system, the total number of adsorption sitesfor a dyestuff or the like can be obtained for each type of layeredinorganic compound, and each type of layered inorganic compound can beadded so that the maximum amount of the dyestuff or the like which canbe formed does not exceed the whole adsorption sites of the layeredinorganic compound.

[0271] As described above, since the degree of adsorption is influencedby the composition (pH, ion strength, complex forming components and thelike) of a buffer, it can be adjusted to a desired degree by changingthe composition, concentration or pH of the buffer, or changing theamount of a compound added which can compete with a dyestuff or the likein adsorption to the layered inorganic compound. Examples of thecompetitive compound include metal ions, organic amines, carboxylicacids, phosphates and the like. Surfactants and soluble polymers canalso be used.

[0272] The type of a buffer or a buffer solution used, the pH andconcentration of the buffer, and the like are the same as thosedescribed in the section of the measuring method of the presentinvention.

[0273] The method of adding a buffer is not particularly limited. Abuffer may be added as a buffer solution having the layered inorganiccompound dissolved or dispersed therein or contained as a dried producttogether with the layered inorganic compound.

[0274] In the production of a testing piece, although a translucentcolloidal agglomerate may be formed in a dispersion of the layeredinorganic compound, this agglomerate is uniformly re-dispersed bystirring the dispersion. When the agglomerate is particularlyinconvenient, it is recommended to use a phosphate buffer solution toimprove the dispersibility of the layered inorganic compound, therebymaking it possible to suppress the formation of the agglomerate.

[0275] Various surfactants can be further contained in the test portion.Addition of a surfactant improves the coating properties of the testportion or the like on the support. Since the surfactant has functionsto adsorb to the interface and disperse or dissolve a substance, itconflicts with the adsorption of the formed detectable substance to thelayered inorganic compound or dissolve the formed detectable substance,whereby the effect of the present invention may be weakened. Therefore,a surfactant which does not interfere the adsorption of the formeddetectable substance to the layered inorganic compound is preferablyselected as the surfactant to be used in combination of the layeredinorganic compound in the present invention. The amount of thesurfactant used is preferably so small as to avoid the aboveinterruption. The type and amount of the surfactant are the same asthose described in the above section of the measuring method of thepresent invention.

EXAMPLES

[0276] The following examples are given to further illustrate thepresent invention.

Example 1

[0277] Peroxidase (POD), 4-AA andN-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline (to beabbreviated as EHSDA hereinafter) as dyestuff precursors, bis-trisbuffer (pH 6.5) as a buffer, and smectite as a layered inorganiccompound were taken to final concentrations shown in. Table 2, andhydrogen peroxide was added to these to a final concentration of 120mmol/l and allowed to react to obtain a reaction solution. Theabsorption spectrum of an agglomerated portion of the obtained reactionsolution was measured at a wavelength of 450 to 750 nm.

[0278] POD, 4-AA, EHSDA and a bis-tris buffer were taken to finalconcentrations shown in Table 3, and hydrogen peroxide was added tothese to a final concentration of 120 mmol/l and allowed to react toobtain a reaction solution. The absorption spectrum of the obtainedreaction solution was measured at a wavelength of 450 to 750 nm as well.

[0279] The measurement of absorbance was carried out using theJascoV-550 (Japan Spectroscopic Co. Ltd.) at intervals of 0.5 nm. Thescanning rate was 200 nm/min and the bandwidth was 1.0 nm. A disposablecell having a cell length of 1 cm (made from polymethyl methacrylate)was used and a 0.1-ml slit was used to measure only an agglomeratedportion as an agglomerate was produced when smectite was added. Theresults of measurement are shown in FIG. 1. TABLE 2 Reagent Finalconcentration POD (peroxidase) 1 U/mil 4-AA *1 2 mmol/l EHSDA *2 2mmol/l Bis-tris buffer *3 100 mmol/l Smectite *4 0.1% (total amount of 3ml)

[0280] TABLE 3 Reagent Final concentration POD 1 U/ml 4-AA 2 mmol/lEHSDA 2 mmol/l Bis-tris buffer 100 mmol/l (total amount of 3 ml)

[0281] The reagents used are shown in Table 4 below. TABLE 4 ReagentReagent concentration Maker Reagent purity POD 30 U/ml Toyobo 4-AA 60mmol/l Wako Chamicals Guaranteed reagent grade EHSDA 60 mmol/l SIGMABis-tris 0.25 mmol/l Nacalaitesque Specially buffer prepared Smectite0.3% Co-op Chemical Hydrogen Santoku Kagaku Guaranteed peroxide Kogyoreagent grade

[0282] As seen from the results of FIG. 1, it could be confirmed that areaction proceeded under the condition that a dyestuff was adsorbed tosmectite like the condition that no smectite was added. The absorptionmaximum when no smectite was added was about 593 nm and the absorptionmaximum when smectite was added was about 578 nm.

Example 2

[0283] POD, 4-AA, EHSDA and a bis-tris buffer (pH 6.5) were taken into aquartz cell having a cell length of 1 cm to final concentrations shownin Table 3 and incubated at 37° C. for 3 minutes. After the temperaturewas adjusted, hydrogen peroxide having a concentration shown in Table 5was added to start a reaction and absorbance was measured 3 minutesafter the start of the reaction. This reaction reached a terminationcompletely 3 minutes after the measurement.

[0284] The instrument used was the JascoV-550 (Japan Spectroscopic Co.Ltd.), and the measurement wavelength was 593 mm (wavelength near theabsorption maximum). A calibration curve of hydrogen peroxide when nosmectite was added could be obtained from this result. TABLE 5Concentration of hydrogen Absorbance peroxide (μmol/l) First time Secondtime Average 200 1.70 1.70 1.70 100 0.85 0.85 0.85 50 0.42 0.42 0.42 250.21 0.21 0.21 13 0.10 0.10 0.10 6.3 0.04 0.04 0.04 4.2 0.01 0.01 0.011.6 0.01 0.01 0.01

Example 3

[0285] <Experimental Method>

[0286] POD, 4-AA, EHSDA, a bis-tris buffer (pH 6.5) and syntheticsmectite were taken into a disposable cell having a cell length of 1 cm(made from polymethyl methacrylate) to final concentrations shown in theabove Table 2, and the temperature was adjusted to 37° C. for 180seconds. After the temperature adjustment, hydrogen peroxide was addedto a final concentration shown in Table 6, and absorbance was measuredfrom 10 seconds after the addition of hydrogen peroxide for 1,800seconds at intervals of 2 seconds. The JascoV-550 (Japan SpectroscopicCo. Ltd.) was used as a measuring device, and the measurement wavelengthwas 577 nm (wavelength near the maximum absorption wavelength). Tomeasure only an agglomerated portion, a 0.1-ml slit was used. Themeasurement result at a hydrogen peroxide concentration of 0 μmol/l wasmade blank, the difference of absorbance (ΔAbs) from the blank at 1,800seconds after the start of measurement was obtained, and a calibrationcurve of hydrogen peroxide when smectite was added was obtained.

[0287] <Result>

[0288] The result is shown in FIG. 2 together with the result when nosmectite was added in Example 2. A calibration curve showing thelogarithmic representation in both of the axis of ordinates and the axisof abscissa of the calibration curve of FIG. 2 is shown in FIG. 3. It isunderstood from Tables 5 and 6 and FIGS. 2 and 3 that there is acorrelation between absorbance resulted by the adsorped dyestuff andhydrogen peroxide. When smectite was added, a calibration curve of r0.999 (r=correlation coefficient) was obtained at a hydrogen peroxideconcentration of 0 to 200 mmol/l.

[0289] It is seen from FIGS. 2 and 3 that when no smectite was added,the minimum detection limit was about 6 mol/l whereas when smectite wasadded, the minimum detection limit was improved to 3 pmol/l. The slopeof the calibration curve was about 2 times that when no smectite wasadded. TABLE 6 Concentration of hydrogen peroxide (μmol/l) AbsorbanceΔAbs 200 3.61 3.30 100 2.02 1.71 50 1.01 0.70 25 0.76 0.45 13 0.49 0.186.3 0.40 0.09 3.1 0.36 0.05 1.6 0.31 0.00 0 0.31

Example 4

[0290] <Experimental Method>

[0291] POD, 4-AA, EHSDA, a bis-tris buffer (pH 6.5) and syntheticsmectite were taken into a disposable cell having a cell length of 1 cm(made from polymethyl methacrylate) to final concentrations shown in theabove Table 2, and the temperature was adjusted to 37° C. for 180seconds. After the temperature adjustment, hydrogen peroxide was addedto a final concentration of 100 μmol/l, and absorbance was measured from20 seconds after the addition of hydrogen peroxide for 600 seconds atintervals of 2 seconds. The JascoV-550 (Japan Spectroscopic Co. Ltd.)was used as a measuring intrument, and the measurement wavelength was577 nm (wavelength near the maximum absorption wavelength). To measureonly an agglomerated portion, a 0.1-ml slit was used. The measurementresult at a hydrogen peroxide concentration of 0 μmol/l was made blank.

[0292] POD, 4-AA, EHSDA and a bis-tris buffer (pH 6.5) were taken into adisposable cell having a cell length of 1 cm (made from polymethylmethacrylate) to final concentrations shown in the above Table 3, andthe temperature was adjusted to 37° C. for 180 seconds. After thetemperature adjustment, hydrogen peroxide was added to a finalconcentration of 100 μmol/l, and absorbance was measured from 20 secondsafter the addition of hydrogen peroxide for 600 seconds at intervals of2 seconds. The JascoV-550 (Japan Spectroscopic Co. Ltd.) was used as ameasuring instrument, and the measurement wavelength was 593 nm(wavelength near the maximum absorption wavelength). A 0.1-ml slit wasused.

[0293] <Results>

[0294] The results are shown in FIG. 4. It is understood from FIG. 4that the addition of smectite is effective in increasing sensitivity. Itis confirmed that a color developing reaction reaches a termination inabout 30 seconds after the addition of hydrogen peroxide. Even whensmectite was added after color was developed without addition ofsmectite, the adsorption and agglomeration of a dyestuff were observed.

Example 5

[0295]3,3′-(3,3′-Dimethoxy-4,4′-bisphenylene)-bis[2-(p-nitrophenyl)-5-phenyl-2H-tetrazoliumchloride (to be abbreviated as “tetrazolium salt” hereinafter) as atetrazolium salt, a phosphate buffer (a mixture of disodiumhydrogenphosphate and sodium dihydrogenphosphate having a pH of 8.5) asa buffer, L-ascorbic acid and smectite (trade name: Lucentite SWN:synthetic smectite manufactured by Co-op Chemical Co.) were taken into adisposable tube to final concentrations shown in Table 7 and allowed toreact with one another to develop color. This reaction is known as areaction for forming water-insoluble formazan. The obtained colordeveloping solution was diluted 10 times, and the absorption spectrum ofthe solution was measured at a wavelength of 400 to 800 nm.

[0296] For comparison, the tetrazolium salt, the phosphate buffer andL-ascorbic acid were taken into a disposable tube to finalconcentrations shown in Table 7 as described above without addition ofsmectite and allowed to react with one another to develop color. Theabsorption spectrum of the obtained color developing solution wasmeasured at a wavelength of 400 to 800 nm.

[0297] The JascoV-550 spectrophotometer of Japan Spectroscopic Co. Ltd.was used to measure absorbance. A disposable cell (made from polymethylmethacrylate) having a cell length of 1 cm was used. The measurementresults are shown in FIG. 5. TABLE 7 Reagent Final concentrationTetrazolium salt 1 mmol/l Phosphate buffer 100 mmol/l L(+)-Ascorbic acid333 μmol/l Smectite 0.1% or 0% (total amount of 3 ml)

[0298] Blue was developed, and the absorption maximum wavelength wasabout 633 nm in the smectite-non-added system. Red purple was developed,and the absorption maximum wavelength was about 535 nm in thesmectite-added system. It could be confirmed that a reaction proceededunder the condition that smectite was added like the condition that nosmectite was added. Further, a deposit considered as formazan separatedout on the inner surface of the cell in the smectite-non-added systemwhereas neither a precipitate nor an agglomerate was observed in thecell in the smectite-added system. Since the absorption maximum shiftsto a short wavelength side, it is understood that when the same colordeveloping reaction system as that of this example is used, it is betterto carry out the measurement of absorbance at 633 nm (for thesmectite-non-added system) and 535 nm (for the smectite-added system)which are wavelengths near the absorption maximum wavelengths of thesmectite-added system and the smectite-non-added system, respectively.

Example 6

[0299] The same tetrazolium salt, phosphate buffer (pH 8.5) and smectiteas those used in Example 5 were taken into a disposable tube to finalconcentrations shown in Table 8 and incubated at 30° C. for 3 minutes.After incubation, L-ascorbic acid was added to final concentrations (0to 333 mmol/l) shown in Table 9 and allowed to react at 30° C. for 30minutes, and absorbance was measured (measurement wavelength of 535 nm).A sample obtained when ascorbic acid was not added was made blank, and acalibration curve was drawn from the measurement results.

[0300] For comparison, the tetrazolium salt and the phosphate bufferwere taken into a disposable tube to final concentrations shown in Table8 in the same manner as described above except that smectite was notadded and incubated at 30° C. for 3 minutes. After incubation, ascorbicacid was added to final concentrations (0 to 333 mol/l) shown in Table10 and allowed to react at 30° C. for 30 minutes, and absorbance wasmeasured (measurement wavelength of 633 nm). A sample obtained whenascorbic acid was not added was made blank, and a calibration curve wasdrawn from the measurement results.

[0301]FIG. 6 shows the obtained calibration curves. In thesmectite-non-added system, a calibration curve of r (correlationcoefficient)=0.9972 was obtained at an ascorbic acid final concentrationof 41.7 to 333.3 μmol/l. In the smectite-added system, a calibrationcurve of r=0.9985 was obtained at an ascorbic acid final concentrationof 5.2 to 133.3 μmol/l. It is seen that the slope of the obtainedcalibration curve in the smectite-added system was about 2.5 times thatof the smectite-non-added system and that a sensitivity improving effectcould be obtained by the addition of smectite. Further, a depositconsidered as formazan separated out on the inner surface of the tube inthe smectite-non-added system whereas neither a precipitate nor anagglomerate was observed in the tube in the smectite-added system.

[0302] The JascoV-550 spectrophotometer of Japan SpectroscopicCo. Ltd.was used to measure absorbance. A disposable cell (made from polymethylmethacrylate) having a cell length of 1 cm was used. TABLE 8 ReagentFinal concentration Tetrazolium salt 800 μmol/l Phosphate buffer (pH8.5) 100 mmol/l L(+)-Ascorbic acid 0-333.3 μmol/l Smectite 0.1% or 0%(total amount of 3 ml)

[0303] TABLE 9 Smectite-added system Final concentration of ascorbicacid (μmol/l) Absorbance (Abs) 5.2 0.174 10.4 0.259 20.8 0.437 41.70.818 66.7 1.165 83.3 1.404 133.3 2.097

[0304] TABLE 10 Smectite-non-added system Final concentration ofascorbic acid (μmol/l) Absorbance (Abs) 41.7 0.103 62.5 0.178 83.3 0.271125.0 0.480 166.7 0.773 250.0 1.358 333.3 1.903

Example 7

[0305] Hydrochloric acid, smectite (Lucentite SWN of Co-op ChemicalCo.), 2,4-dichloroaniline and sodium nitrite were taken to finalconcentrations shown in Table 11 in the mentioned order and mixedtogether, and a Tsuda reagent(N,N-diethyl-N′-1-naphthylnaphthylethylene-diamine oxalate) was addedand allowed to react with the mixture. Thus, an azo dyestuff was formedand caused to develop color. The absorption spectrum of this dyestuffwas measured at a wavelength of 400 to 800 nm. The sodium nitrite wasadded in four different concentrations (0, 8, 16 and 33 mmol/l). Theresults are shown in FIG. 7.

[0306] For comparison, hydrochloric acid, 2,4-dichloroaniline and sodiumnitrite were added to final concentrations shown in Table 11 in the samemanner as described above except that smectite was not added, and aTsuda reagent was added to develop color. The absorption spectrum of theresulting mixture was measured at a wavelength of 400 to 800 nm. Thesodium nitrite was added in four different concentrations (0, 8, 16 and33 mmol/l). The results are shown in FIG. 8. The absorption spectra ofthe smectite-added system and the smectite-non-added system obtainedwhen the concentration of sodium nitrite was 33 μmol/l are shown in FIG.9. The JascoV-550 spectrophotometer of Japan Spectroscopic Co. Ltd. wasused to measure absorbance. A disposable cell (made form polymethylmethacrylate) having a cell length of 1 cm was used. TABLE 11 ReagentFinal concentration Hydrochloric acid 1 mol/l 2,4-Dichloroaniline 200μmol/l Sodium nitrite 0-33 μmol/l Tsuda reagent 200 μmol/l Smectite 0.1%or 0% (total amount of 3 ml)

[0307] In the smectite-non-added system, red purple was developed andthe absorption maximum wavelength was about 540 nm. In thesmectite-added system, purple was developed and the absorption maximumwavelength was about 555 nm. It could be confirmed that a reactionproceeded under the condition that smectite was added like the conditionthat no smectite was added. Since the absorption maximum shifts to along wavelength side, it is understood that when the same colordeveloping reaction system as that of this example is used, it is betterto carry out the measurement of absorbance at 540 nm (for thesmectite-non-added system) and 555 nm (for the smectite-added system)which are wavelengths near the absorption maximum wavelengths of thesmectite-added system and the smectite-non-added system, respectively.

Example 8

[0308] The same hydrochloric acid, 2,4-dichloroaniline, sodium nitriteand Tsuda reagent as those used in Example 7 were taken into adisposable cell (made from polymethyl methacrylate) to finalconcentrations shown in Table 12 and allowed to react with one anotherat 30° C. for 10 minutes. After color was fully developed, smectite wasadded to form and precipitate an agglomerate. From 30 seconds after theaddition of smectite, the absorbance of the agglomerate was measured for20 minutes at intervals of 1 second (measurement wavelength of 555 nm).To measure only the absorbance of the agglomerate, a 0.1-ml slit wasused. Sodium nitrite was added to final concentrations shown in Table 13(0 to 50 μmol/l). The result of a sample obtained when the finalconcentration of sodium nitrite was 0 μmol/l was made blank, and thedifference of absorbance from the blank (ΔAbs) 20 minutes after thestart of measurement was obtained to draw a calibration curve.

[0309] As a smectite-non-added system, hydrochloric acid,2,4-dichloroaniline and sodium nitrite were taken into a disposable cellto final concentrations shown in Table 12 in the same manner asdescribed above and incubated at 30° C. for 3 minutes. A Tsuda reagentwas added, and absorbance was measured from 10 seconds after theaddition for 10 minutes at intervals of 1 second (measurement wavelengthof 540 nm). Sodium nitrite was added to final concentrations shown inTable 14 (0 to 50 μmol/l). Absorbance 10 minutes after the start ofmeasurement was obtained to draw a calibration curve. The result of asample obtained when the final concentration of sodium nitrite was 0μmol/l was made blank. The reaction system used in this experimentreached a termination completely in 10 minutes. FIG. 10 shows theobtained calibration curves. In the smectite-non-added system, acalibration curve of r (correlation coefficient)=0.9991 was obtained ata sodium nitrite final concentration of 1.6 to 50.0 μmol/l. In thesmectite-added system, a calibration curve of r 0.9940 was obtained at asodium nitrite final concentration of 0.4 to 25.0 μmol/1. Theinclination of the obtained calibration curve in the smectite-addedsystem was about 2.5 times that of the smectite-non-added system. It isunderstood that a sensitivity increasing effect can be obtained by theaddition of smectite.

[0310] The JascoV-550 spectrophotometer of Japan Spectroscopic Co. Ltd.was used to measure absorbance. A disposable cell (made from polymethylmethacrylate) having a cell length of 1 cm was used. TABLE 12 ReagentFinal concentration Hydrochloric acid  1 μmol/l 2,4-Dichloroaniline 200μmol/l Sodium nitrite 0-50 μmol/l  Tsuda reagent 200 μmol/l Smectite0.1% or 0% (total amount of 3 ml)

[0311] TABLE 13 Smectite-added system Final concentration of sodiumnitrite (μmol/l) Absorbance (ΔAbs) 0.4 0.099 0.8 0.161 3.1 0.445 12.51.610 20.0 2.034 25.0 2.620

[0312] TABLE 14 Smectite-non-added system Final concentration of sodiumnitrite (μmol/l) Absorbance (Abs) 1.6 0.103 6.3 0.178 12.5 0.271 25.00.480 50.0 1.358

Example 9

[0313] POD, 4-AA andN-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline (to beabbreviated as EHSDMeA hereinafter) as dyestuff precursors, a bis-trisbuffer (pH 6.5) as a buffer and smectite as a layered inorganic compoundwere taken into a disposable cell (made from methacrylate) having a celllength of 1 cm to final concentrations shown in Table 15, and thissample was incubated at 37° C. for 180 seconds. As another sample, theabove components excluding smectite were prepared to the sameconcentrations and incubated likewise.

[0314] After temperature adjustment, hydrogen peroxide was added to eachof these samples to a final concentration shown in Table 15, andabsorbance was measured from 20 seconds after the addition of hydrogenperoxide for 1,800 seconds at intervals of 2 seconds. The JascoV-550spectrophotometer of Japan Spectroscopic Co. Ltd. was used, and themeasurement wavelength was 630 nm. Since an agglomerate was formed whensmectite was added, a 0.1-ml slit was used to measure only anagglomerated portion. As a background for the smectite-added system, asample was prepared by adding smectite to the same concentration withoutadding hydrogen peroxide, and this reaction solution was measuredlikewise. TABLE 15 Reagent Final concentration POD 1 U/mL 4-AA*1 0.05mmol/L EHSDMeA*2 5 mmol/L Bis-tris buffer*3 100 mmol/L Smectite 0.1%Hydrogen peroxide 100 mmol/L (total amount of 3 ml)

[0315] The used reagents are shown in Table 16 below. TABLE 16 ReagentReagent concentration Maker Reagent purity POD 30 U/mL Toyobo 4-AA 1.5mmol/L Wako Chemicals Guaranteed reagent grade EHSDMeA 150 mmol/L DojinKagaku Bis-tris 0.25 mmol/L Nacalaitesque Specially buffer preparedSmectite 0.3% Co-op Chemical Hydrogen 300 mmol/L Santoku KagakuGuaranteed peroxide Kogyo reagent grade

[0316] The results are shown in FIG. 11. As seen from FIG. 11, areduction in absorbance was observed about 3 minutes after the start ofa reaction in the smectite-non-added system whereas no reduction inabsorbance was observed in the smectite-added system. Therefore, itcould be confirmed that the detection reaction proceeded when smectitewas added like when no smectite was added and further that a dyestuffformed by an oxidation condensation between 4-AA and EHSDMeA was notoxidized and decomposed by hydrogen peroxide and its discoloration wassuppressed due to its adsorption to smectite.

Example 10

[0317] POD, 4-AA and EHSDMeA, a bis-tris buffer (pH 6.5) and smectitewere taken into a disposable cell (made from methacrylate) having a celllength of 1 cm to final concentrations shown in Table 17 and Table 18 toprepare five different samples (Sample Nos. 1 to 5), and each sample wasincubated at 37° C. for 180 seconds.

[0318] After temperature adjustment, hydrogen peroxide was added to eachof these samples in an amount shown in Table 18 to start a reaction.Ascorbic acid was also added in an amount shown in Table 18, 60 secondsafter the addition of hydrogen peroxide, and absorbance was measured 20seconds after the addition for 300 seconds at intervals of 1 seconds.The JascoV-550 spectrophotometer of Japan Spectroscopic Co. Ltd. wasused as a measuring device, and the measurement wavelength was 630 nm. A0.1-ml slit was used to measure only an agglomerated portion. TABLE 17Reagent Final concentration POD 1 U/mL 4-AA 2 mmol/L EHSDMeA 2 mmol/LBis-tris buffer 100 mmol/L Smectite*1 (see Table 18) Hydrogen peroxide(see Table 18) L(+)-Ascorbic acid (see Table 18) (total amount of 3 ml)

[0319] TABLE 18 Smectite Ascorbic acid Hydrogen peroxide Sample No. (%)(mg/dl) (μmol/l) 1 0 0 100 2 0 5 (284 μmol/L) 100 3 0.1 0 100 4 0.1 5(284 μmol/L) 100 5 0.1 0 0

[0320] The used reagents are shown in Table 19 below. TABLE 19 ReagentReagent concentration Maker Reagent purity POD 30 U/mL Toyobo 4-AA 60mmol/L Wako Chemicals Guaranteed reagent grade EHSDAMeA 60 mmol/L DojinKagaku Bis-tris 0.25 mmol/L Nacalaitesque Specially buffer preparedSmectite 0.3% Co-op Chemical Hydrogen 3 mmol/L Santoku Kagaku Guaranteedperoxide Kogyo reagent grade Ascorbic 150 mg/dl Nacalaitesque Guaranteedacid reagent grade

[0321] The results are shown in Table 18 and FIG. 12. FIG. 13 is anenlarged view of a section for 0 to 60 seconds in FIG. 12. Absorbances(Abs) 0, 60 and 300 seconds after the start of measurement are shown inTable 20. Further, to subtract the influence of agglomeration, ΔAbs wasobtained for Sample Nos. 3 and 4 from the difference from the absorbanceof Sample No. 5. The results are shown as data within parentheses inTable 20.

[0322] As seen from FIGS. 12 and 13, in the smectite-non-added system,discoloration occurred immediately after ascorbic acid was added. At thetime of the start of measurement (20 seconds after the addition ofascorbic acid), only about 20% of color development when ascorbic acidwas not added was seen and the sample became achromatic 60 seconds afterthe start of measurement (80 seconds after the addition of ascorbicacid).

[0323] To contrary, in the smectite-added system, about 90% of colordevelopment was seen at the time of the start of measurement (20 secondsafter the addition of ascorbic acid), about 80% of color development wasseen 60 seconds after the start of measurement (80 seconds after theaddition of ascorbic acid), and about 50% of color development was seen300 seconds after the start of measurement (320 seconds after theaddition of ascorbic acid).

[0324] It could be confirmed from this result that the reduction anddecomposition of a formed dyestuff by ascorbic acid was suppressed bythe addition of smectite. TABLE 20 Sample 0 second 60 seconds 300seconds No. after (ΔAbs) after (ΔAbs) after (ΔAbs) 1 1.15 1.14 1.11 20.23 0.00 0.00 3 1.26 (1.16) 1.33 (1.23) 1.63 (1.49) 4 1.16 (1.06) 1.07(0.97) 0.84 (0.70) 5 0.10 0.10 0.14

Example 11

[0325]3,3′-(3,3′-Dimethoxy-4,4′-biphenylene)-bis[2-(p-nitrophenyl)-5-phenyl-2H-tetrazolium]chloride(to be abbreviated as “tetrazolium salt” hereinafter) as a tetrazoliumsalt, a phosphate buffer (a mixture of disodium hydrogenphosphate andsodium dihydrogenphosphate having a pH of 8.5) as a buffer, L-ascorbicacid and smectite (trade name: Lucentite SWN, synthetic smectitemanufactured by Co-op Chemical Co.) were taken into a disposable cell(made from polymethyl methacrylate) to final concentrations shown inTable 21 and incubated at 30° C. for 180 seconds. Thereafter, L-ascorbicacid was added to start a reaction. Absorbance was measured from 10seconds after addition for 300 seconds at intervals of 1 second toobserve time-cource of absorbance. The measurement wavelength was 535nm, and the reaction temperature was 30° C.

[0326] For comparison, the tetrazolium salt and the phosphate bufferwere taken into a disposable cell (made from polymethyl methacrylate) tofinal concentrations shown in Table 21 in the same manner as describedabove except that smectite was not added and incubated at 30° C. for 180seconds. Thereafter, L-ascorbic acid was added to start a reaction.Absorbance was measured from 10 seconds after addition for 300 secondsat intervals of 1 second to observe time-cource of absorbance. Themeasurement wavelength was 633 nm, and the reaction temperature was 30°C.

[0327] The JascoV-550 spectrophotometer of Japan Spectroscopic Co. Ltd.was used to measure absorbance, and a disposable cell (made frompolymethyl methacrylate) having a cell length of 1 cm was used. Both ofthe above measurement wavelengths are near the absorption maximumwavelengths. FIG. 14 is a graph showing absorbance with respect toelapsed time. The reaction became stable about 50 seconds after thestart of measurement in the smectite-added system whereas the reactionbecame stable about 300 seconds after the start of measurement in thesmectite-non-added system. It is understood from this that the reactionrate is increased by the addition of smectite.

[0328] This reaction is known as a reaction for forming water-insolubleformazan. In the smectite-added system, precipitation or agglomerationwas not observed in the cell. TABLE 21 Reagent Final concentrationTetrazolium salt 800 μmol/l Phosphate buffer (pH 8.5) 100 mmol/lL(+)-ascorbic acid 83.3 μmol/l Smectite 0.1% or 0% (total amount of 3ml)

Example 12

[0329] Hydrochloric acid, smectite (Lucentite SWN of Co-op ChemicalCo.), 2,4-dichloroaniline and sodium nitrite were taken into adisposable cell (made from polymethyl methacrylate) to finalconcentrations shown in Table 22 and incubated at 30° C. for 180seconds. The concentration of sodium nitrite was 1.6, 6.3, 12.5, 35.0and 50.0 mmol/l within the range of 0 to 50 mmol/l.

[0330] After incubation, a Tsuda reagent(N,N-diethyl-N′-1-naphthylnaphthylethylenediamine oxalate) was added,and absorbance was measured from 10 seconds after addition for 600seconds at intervals of 1 second to observe time-cource of absorbance.The measurement wavelength was 555 nm, and the reaction temperature was30° C.

[0331] For comparison, hydrochloric acid, 2,4-dichloroaniline and sodiumnitrite were taken into a disposable cell (made from polymethylmethacrylate) to final concentrations shown in Table 22 in the samemanner as described above except that smectite was not added andincubated at 30° C. for 180 seconds. Thereafter, a Tsuda reagent wasadded, and absorbance was measured from 10 seconds after addition for600 seconds at intervals of 1 second to observe time-cource ofabsorbance. The measurement wavelength was 540 nm, and the reactiontemperature was 30° C.

[0332] The JascoV-550 spectrophotometer of Japan Spectroscopic Co. Ltd.was used to measure absorbance, and a disposable cell (made frompolymethyl methacrylate) having a cell length of 1 cm was used. Both ofthe measurement wavelengths are near the absorption maximum wavelengths.

[0333] FIGS. 15 to 17 are graphs showing the measurement results ofabsorbance with respect to elapsed time. Out of these, FIG. 15 is agraph in the smectite-non-added system, FIG. 16 is a graph in thesmectite-added system, FIG. 17 is a graph in the smectite-added systemand smectite-non-added system when the concentration of sodium nitriteis 25.0 μmol/l. According to these figures, the reaction reaches atermination in about 300 seconds after the start of measurement in thesmectite-non-added system whereas the reaction reaches a termination inabout 30 seconds after the start of measurement in the smectite-addedsystem. It is understood from this that the reaction rate is increasedby the addition of smectite. TABLE 22 Reagent Final concentrationHydrochloric acid 1 mol/l 2,4-Dichloroaniline 200 μmol/l Sodium nitrite0-50 μmol/l Tsuda reagent 200 μmol/l Smectite 0.1% or 0% (total amountof 3 ml)

Example 13

[0334] POD, 4-AA andN-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxylaniline (to beabbreviated as EHSDA hereinafter) as dyestuff precursors and a bis-trisbuffer (pH 6.5) as a buffer were taken to final concentrations shown inTable 23, and hydrogen peroxide was added to these to obtain a colordeveloping solution. 30 μl of the obtained color developing solution wasdropped onto filter paper (No.2 of Toyo Roshi Co.) which was impregnatedwith a 1% dispersion (solvent: distilled water) of a layered inorganiccompound (synthetic smectite: trade name Lucentite SWN of Co-op ChemicalCo.) and dried, and untreated filter paper to observe the diffusion ofthe color developing solution. Assuming that the color developingsolution was infiltrated and diffused in a circle, the maximum portionand the minimum portion of the diameter of the circle were measured toobtain the area of a dyestuff-diffused spot from a mean value of these.TABLE 23 Reagent Final concentration POD 1 U/mL 4-AA *1 2 mmol/L EHSDA*2 2 mmol/L Bis-tris buffer solution *3 100 mmol/L Hydrogen peroxide 100μmol/L (total amount of 3 ml)

[0335] The used reagents are shown in Table 24 below. TABLE 24 ReagentReagent concentration Maker Reagent purity POD 30 U/mL Toyobo 4-AA 60mmol/L Wako Chemials Guaranteed reagent grade EHSDA 60 mmol/L SIGMABis-tris 0.25 mmol/L Nacalaitesque Specially buffer prepared Smectite 1%Co-op Chemical Hydrogen Santoku Kagaku Guaranteed peroxide Kogyo reagentgrade

[0336] The obtained diameter, area and color tone of the spot are shownin Table 25. FIG. 18 and FIG. 19 schematically show filter paper inwhich the color developing solution is diffused. TABLE 25 Diameter Area(mm) (mm²) Color Filter paper impregnated 7-8 44 Blue with smectitepurple Untreated filter paper 20-22 350 Blue

[0337] In the filter paper impregnated with a dispersion of smectite,the diffusion of a dyestuff was suppressed more and the spot was smaller(about ⅛ in area) compared with the untreated filter paper. However, anachromatic portion devoid of a dyestuff of the color developing solutionwas diffused to the same extent as that of the untreated filter paper.It was confirmed from this that the dyestuff was selectively adsorbed tosmectite contained in the filter paper.

[0338] It was observed visually that the color tone of the spot wasdarker in the filter paper impregnated with smectite than in theuntreated filter paper and that the color tone shifted to a shortwavelength side. Further, even when the filter paper impregnated withsmectite was washed with water, the dyestuff did not elute.

[0339] It was found that the dyestuff was adsorbed to the filter paperby the addition of smectite to the filter paper and that the diffusionof the dyestuff was thereby prevented and the elution of the dyestuffwas also prevented.

[0340] Therefore, it is understood that, when the testing piece of thepresent invention is used, the formed dyestuff does not move or eluteand the accuracy and sensitivity of measurement can be improved. It isalso understood that measurement can be carried out with ease becausethe dyestuff is not moved or concentrated by the drying of the testportion and the formed dyestuff does not elute even if the testing pieceis kept immersed in the sample.

Example 14

[0341] Urine test paper (multi-item test paper for measuring nitrite,glucose, occult blood, bilirubin and urobilinogen contained in theurine: general test paper available on the market, prepared by forming atest portion by impregnating filter paper with each reagent and affixingthe filter paper to a plastic film together with a test portion forcalibration) was immersed in control urine prepared by generalformulation, pulled up immediately and left to stand for about 30seconds until coloration was observed, and a piece of the samesmectite-impregnated filter paper as used in Example 13 was pressedagainst the test paper to transfer a dyestuff to the piece of thesmectite-impregnated filter paper. The spreading of the dyestuff on thepiece of the filter paper was observed visually. The piece of the filterpaper was washed well with running water to observe visually whethercolor came out.

[0342] As controls, Toyo Filter Paper No.2 and Toyo Filter Paper No. 131not treated with smectite were used to carry out the same operation.

[0343] The measuring method and formulation in each test are as follows.The results are shown in Table 26 below.

[0344] Nitrite Test: Griess Method

[0345] 4-Aminobenzenearsonic acid was allowed to react with a nitriteunder an acidic condition to form a diazonium salt and the diazoniumsalt was then coupled with N-1-naphthylethylenediamine dihydrochlorideto form an azo dyestuff. As for formulation, one sheet of filter paperwas impregnated with 0.26 mg of N-1-naphthylethylenediaminedihydrochloride and 0.57 mg of 4-aminobenzenearsonic acid and dividedinto 100 portions. One of them was made a test portion. One portionabsorbed about 6 μl of a solution.

[0346] Glucose Test:

[0347] Hydrogen peroxide formed by glucose oxidase was allowed to reactwith a color indicator (tetrabase and guaiac as chromogens) by thecatalytic function of peroxidase to develop color by oxidation. As forformulation, one sheet of filter paper was impregnated with 470 IU ofglucose oxidase, 219 PU of peroxidase, 13.0 mg of tetrabase and 4.3 mgof guaiac and divided into 100 portions. One of them was made a testportion. One portion absorbed about 6 μl of a solution.

[0348] Occult Blood Test:

[0349] This is a method making use of the decomposition of cumenehydroperoxide by hemoglobin and the oxidation color development ofo-tolidine by the oxygen of a formed active group. The same effect canbe expected when a benzidine (such as 3,3′,5,5′-tetramethyl-benzidine)is used in place of o-tolidine. As for formulation, one sheet of filterpaper was impregnated with 52.6 mg of cumene hydroperoxide and 7.6 mg ofo-tolidine and divided into 100 portions. One of them was made a testportion. One portion absorbed about 6 μl of a solution.

[0350] Bilirubin Test:

[0351] This is a method making use of a reaction in which a diazoniumsalt was formed from 2-methyl-5-nitroaniline or sulfanilic acid andsodium nitrite as a diazo reagent under an acidic condition and coupledwith bilirubin in the presence of dyphylline to form azobilirubin. Asfor formulation, one sheet of filter paper was impregnated with 3.8 mgof 2-methyl-5-nitroaniline, 2.1 mg of sodium nitrite and a small amountof dyphylline and divided into 100 portions. One of them was made a testportion. One portion absorbed about 6 μl of a solution.

[0352] Urobilinogen Test:

[0353] This is a method making use of an azo-coupling reaction betweenurobilinogen and a 3,3′-dimethoxybiphenyl-4,4′-diazonium borontetrafluoride salt under an acidic condition. As for formulation, onesheet of filter paper was impregnated with 0.36 mg of3,3′-dimethoxybiphenyl-4,4′-diazonium tetrafluoride borate and dividedinto 100 portions. One of them was made a test portion. One portionabsorbed about 6 μl of a solution. TABLE 26 Spreading of Coming-out ofcolor dyestuff by washing Smectite Untreated Smectite Untreated impreg-No. No. impreg- No. No. Test nated 2 131 nated 2 131 Nitrite test ◯ X X◯ X X Glucose test ◯ X X ◯ X X Occult blood ◯ X X ◯ X X test Bilirubin ◯X X ◯ X X test Urobilinogen ◯ X X ◯ X X test

[0354] As shown by the results of Table 26, a dyestuff was adsorbed tofilter paper impregnated with a layered inorganic compound withoutspeading and color did not come out by washing. Therefore, it isunderstood that the diffusion of the dyestuff is suppressed and theelution of the dyestuff is prevented in the filter paper impregnatedwith the layered inorganic compound. Therefore, in the testing piece ofthe present invention, the formed dyestuff does not move or elute andimprovement in the accuracy and sensitivity of measurement can beexpected. Since the concentration and movement of the dyestuff by dryingthe test portion do not occur and the formed dyestuff does not elutewhile the testing piece is kept immersed in the sample, measurement canbe carried out with ease. Further, since the formed dyestuff does notpollute the adjacent test portion in the multi-item testing piece, theinterval between adjacent test portions is reduced, thereby making itpossible to reduce the size of the testing piece.

[0355] When the formed dyestuff was exposed to the air at roomtemperature without shielding light while the dyestuff was infiltratedinto untreated filter paper, color change and discoloration wereobserved and coloration completely different from that right after areaction was seen about 1 month later. On the other hand, even when theformed dyestuff adsorbed to filter paper containing a layered inorganiccompound was exposed to the air at room temperature without shieldinglight, color change and discoloration were not observed for at least 3months.

[0356] The above facts show the applicability of the present invention.That is, when the testing piece of the present invention is used, asample is taken at a patient's home, reaction coloration is caused onthe testing piece, and this testing piece is mailed to an examinationcenter at a remote place, the same measurement result as that rightafter reaction coloration can be obtained. In other words, the testingpiece of the present invention is stable in coloration, is free from theconcentration of a dyestuff caused by drying and the elution thereofcaused by water leakage. Therefore, it can be used as a mailable testingpiece.

Example 15

[0357] A solution prepared as shown in Table 27 below was applied to apolyethylene terephthalate (PET) film treated with ultraviolet light bya doctor knife to a film thickness of 100 μm and dried. This coatingfilm was cut into a 1-cm square piece together with the PET film, andthe piece was sandwiched between glass plates with 500-μm spacingtherebetween as shown in FIG. 20 to prepare a reaction cell. FIG. 20schematically shows this reaction cell.

[0358] 2 mmol/l of hydrogen peroxide was added to this reaction cell,and color development at this point was observed. A solution prepared inthe same manner as described above except smectite was not added wasused to form a reaction cell, and color development was observed. TABLE27 Reagent Final concentration POD 1 U/mL 4-AA 2 mmol/L EHSDA 2 mmol/LBis-tris buffer solution 100 mmol/L Smectite *1 0.3 HPC-M *2 1%

[0359] The elution of the formed dyestuff from the coating film formedwithout addition of smectite was observed. On the other hand, whensmectite was added, the elution of the formed dyestuff was not observed.

Example 16

[0360] An example of formulation of fabricating the testing piece of thepresent invention having a detection layer composed of a porousstructure is shown below. This testing piece is schematically shown inFIG. 21.

[0361] Filter paper (2 Chr of Whatman Co.) was immersed in a reagentsolution containing GOD (glucose oxidase) and POD as enzymes prepared asshown in Table 28 below and dried at 40° C. for −30 minutes. This filterpaper was cut into a 5 mm×5 mm piece which was then bonded to one end ofa 5 mm×100 mm white plastic film with adhesive double-coated tape toprepare a testing piece having a test portion composed of the filterpaper. TABLE 28 Reagent Final concentration GOD 100 U/mL POD 100 U/mL4-AA 5 g/L EHSDA 3 g/L Phosphate buffer solution (pH 7.0) 0.1 mol/LSmectite 1%

[0362] In this testing piece, 6 Al of blood plasma was dropped onto thetest portion by a pipette, or the testing piece was immersed in theurine collected into a glass, and a reaction was allowed to proceed, andthen the intensity of color developed in the detection layer wasmeasured with a reflectiometer or the like so that the concentration ofglucose contained in the blood plasma or urine can be measured. Theporous structure layer containing the layered inorganic compound in thepresent invention can be used as the detection layer which also servesas the sample suction layer, the reagent layer and the reaction layer inthe testing piece of the present invention.

Example 17

[0363] An example of a method for producing the testing piece having aporous structure detection area of the present invention will bedescribed below. FIG. 22 schematically shows this testing piece.

[0364] Filter paper (2 Chr of Whatman Co.) was immersed in a reagentsolution containing GOD and POD as enzymes prepared as shown in Table 29below and dried at 40° C. for 30 minutes. This filter paper was cut intoa 5 mm×5 mm piece which was then bonded to another 5 mm×100 mm filterpaper (2 Chr of Whatman Co.) at a predetermined location (reaction areain FIG. 22) by pressure. Thereafter, another filter paper (2 Chr ofWhatman Co.) was immersed in a dispersion of a layered inorganiccompound prepared as shown in Table 30 below and dried naturally at roomtemperature. This filter paper was cut into a 5 mm×5 mm piece which wasthen bonded to the above 5 mm×100 mm filter paper (2 Chr of Whatman Co.)having a reaction area at a predetermined location (holding area in FIG.22) by pressure. The thus produced testing piece had a sample suctionarea, a diffusion area, a reaction area, a holding area for adsorbing adetectable substance and an area for absorbing excess of the sample. Theholding area also served as a detection area. TABLE 29 Reagent Finalconcentration GOD 100 U/mL POD 200 U/mL 4-AA 5 g/L EHSDA 3 g/L Phosphatebuffer solution (pH 7.0) 0.1 mol/L

[0365] TABLE 30 Reagent Final concentration Bis-tris buffer 0.1 mol/Lsolution (pH 6.5) Smectite 1%

[0366] The sample suction area of this testing piece was immersed in theblood plasma collected into a cuvette or the urine collected into aglass. The sample passed through the sample suction area and thediffusion area and reached the reaction area where it was mixed with thereagent to become a reaction solution. After the reaction solutionfurther passed through the reaction time control area and the holdingarea, the testing piece was pulled up. The intensity of coloration inthe holding area was measured with a reflectiometer or the like tomeasure the concentration of glucose contained in the blood plasma orthe urine.

[0367] The porous structure containing the layered inorganic compound inthe present invention can be used as the detection area which alsoserves as the holding area for adsorbing a detectable substance(dyestuff) contained in the reaction solution in this example of thetesting piece.

INDUSTRIAL APPLICABILITY

[0368] The measuring method of the present invention can be used as amethod for measuring a substance with high sensitivity and highaccuracy. That is, according to the first method of the presentinvention, high-sensitivity measurement is made possible by measuring adetectable substance after a layered inorganic compound such as a claymineral is added to a reaction system to adsorbe the detectablesubstance. According to the second measuring method of the presentinvention, by adding a layered inorganic compound such as a clay mineralto a reaction system, a detectable substance such as a dyestuff isadsorbed to the layered inorganic compound and protected, whereby thedecomposition of the detectable substance by excess of hydrogenperoxide, reducing ascorbic acid or the like can be suppressed and thedetectable substance can be stabilized. Therefore, the discoloration orthe like can be prevented if a dyestuff is the detectable substance, andstable high-sensitivity and high-accuracy measurement is possible.According to the third method of the present invention, by adding alayered inorganic compound such as a clay mineral to a reaction systemwhich forms a detectable substance, the rate of the formation reactionis increased to enable quick measurement. According to the fourthmeasuring method of the present invention, by carrying out the formationreaction of a detectable substance by dispersing a layered inorganiccompound such as a clay mineral into a reaction solvent,high-sensitivity measurement is made possible even in a reaction systemwhich forms an insoluble substance. According to the testing piece ofthe present invention, a dyestuff or the like is hardly diffused andeluted, and more sensitive and accurate simple analysis is madepossible.

[0369] The measuring method of the present invention can be used for thedetection, determination or the like of bio-components contained in thebody fluid such as urine and blood, foods, medicines, substancesexistent in trace amounts in natural environment, industrial chemicalsubstances, substances contained in trace amounts in waste, and thelike.

1. An analytical testing piece for measuring an analyte by measuring adetectable substance formed by a reaction of the analyte contained in asample with a reagent, wherein the testing piece comprises at least onetest portion having a detection area for detecting the detectablesubstance and a reaction portion where the analyte contained in thesample reacts with the reagent, and the detectable substance is formedin the reaction portion, and the testing piece contains a layeredinorganic compound at least in the test portion.
 2. The testing pieceaccording to claim 1, wherein the test portion comprises a time controlportion for adjusting the proceeding of a reaction making use of a timeduring which the sample moves so that the detectable substance passesthrough the time control portion and reaches the detection area.
 3. Thetesting piece according to claim 1, wherein the test portion has adiffusion area for diffusing the sample so that the sample passesthrough the diffusion area to be diffused and reaches the detectionarea.
 4. The testing piece according to claim 1, wherein the detectionarea is composed of two or more layers including a detection layer fordetecting the detectable substance.
 5. The testing piece according toclaim 1, wherein the detection area is provided at a location which thesample reaches after the sample is diffused and passes through thereaction portion.
 6. The testing piece according to claim 1, wherein aportion containing the layered inorganic compound in the testing pieceis composed of a porous structure.
 7. The testing piece according toclaim 6, wherein the porous structure is formed by the layered inorganiccompound or at least one member selected from the group consisting of ahydrophilic polymer, a membrane filter, a fiber assembly and organic orinorganic compound fine powders.
 8. The testing piece according to claim1, wherein the layered inorganic compound is a 2:1 type clay mineral. 9.The testing piece according to claim 8, wherein the 2:1 type claymineral is a swelling layered clay mineral.
 10. The testing pieceaccording to claim 9, wherein the swelling layered clay mineral is atleast one member selected from the group consisting of bentonite,smectite, vermicuilite and synthetic fluorine mica.
 11. The testingpiece according to claim 10, wherein the smectite is synthetic smectite.12. The testing piece according to claim 11, wherein the syntheticsmectite is at least one member selected from the group consisting ofhectorite and saponite.
 13. The testing piece according to claim 1,wherein the reagent is contained in the test portion by adding asolution of the reagent before and/or after the addition of the sampleto the test portion.
 14. The testing piece according to claim 1, whereina buffer or a dried product thereof is further contained in a portioncontaining the layered inorganic compound.
 15. The testing pieceaccording to claim 1, wherein the reagent is capable of forming asubstance detectable by an optical method when the reagent reacts withthe analyte.
 16. The testing piece according to claim 15, wherein thesubstance detectable by the optical method is water-soluble.
 17. Ananalytical testing piece for measuring an analyte by measuring adetectable substance formed by a reaction of the analyte contained in asample with a reagent, wherein the testing piece comprises at least onetest portion having a detection area for detecting the detectablesubstance and a diffusion area for diffusing the sample so that thesample passes through the diffusion area to be diffused and reaches thedetection area, and the testing piece contains a layered inorganiccompound at least in the test portion.